Pentose fermentation by a recombinant microorganism

ABSTRACT

The present invention provides methods and compositions suitable for use in the conversion of xylose to xylitol and xylulose, including nucleic acid constructs, recombinant fungal host cells, and related materials.

The present application is a Divisional of U.S. patent application Ser. No. 13/429,710, filed on Mar. 26, 2012, now U.S. Pat. No. 8,663,962, and also claims priority to U.S. Pat. Appln. Ser. No. 61/469,505, filed on Mar. 30, 2011, and U.S. Pat. Appln. Ser. No. 61/496,152, filed on Jun. 13, 2011, each of which is incorporated by reference in their entirety herein.

FIELD OF THE INVENTION

The present invention provides methods and compositions suitable for use in the conversion of xylose to xylitol and xylulose, including nucleic acid constructs, recombinant fungal host cells, and related materials.

BACKGROUND

Ethanol and ethanol fuel blends are widely used in Brazil and in the United States as a transportation fuel. Combustion of these fuels is believed to produce fewer of the harmful exhaust emissions (e.g., hydrocarbons, nitrogen oxide, and volatile organic compounds [VOCs]) that are generated by the combustion of petroleum. Bioethanol is a particularly favored form of ethanol because the plant biomass from which it is produced utilizes sunlight, an energy source that is renewable. In the United States, ethanol is used in gasoline blends that are from 5% to 85% ethanol. Blends of up to 10% ethanol (E10) are approved for use in all gasoline vehicles in the U.S. and blends of up to 85% ethanol (E85) can be utilized in specially engineered flexible-fuel vehicles (FFV). The Brazilian government has mandated the use of ethanol-gasoline blends as a vehicle fuel, and the mandatory blend has been 25% ethanol (E25) since 2007.

Bioethanol is currently produced by the fermentation of hexose sugars that are obtained from carbon feedstocks. Currently, only the sugar from sugar cane and starch from feedstock such as corn can be economically converted. There is, however, much interest in using lignocellulosic feedstocks where the cellulose part of a plant is broken down to sugars and subsequently converted to ethanol. Lignocellulosic biomass is made up of cellulose, hemicelluloses, and lignin. Cellulose and hemicellulose can be hydrolyzed in a saccharification process to sugars that can be subsequently converted to ethanol via fermentation. The major fermentable sugars from lignocelluloses are glucose and xylose. For economical ethanol yields, a strain that can effectively convert all the major sugars present in cellulosic feedstock would be highly desirable.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions suitable for use in the conversion of xylose to xylitol and xylulose, including nucleic acid constructs, recombinant fungal host cells, and related materials.

In some embodiments, the present invention provides recombinant nucleic acid constructs comprising at least one polynucleotide sequence that encodes at least one xylose reductase, wherein the polynucleotide is selected from: (a) a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least about 70% identity to SEQ ID NO:2, wherein the amino acid sequence comprises at least one substitution set forth herein; and (b) a polynucleotide that hybridizes under stringent hybridization conditions to the complement of a polynucleotide that encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, and wherein the amino acid sequence comprises at least one substitution set forth herein. In some embodiments, the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:2, wherein the amino acid sequence comprises at least one substitution set forth herein. In some alternative embodiments, the polynucleotide sequence encodes a polypeptide comprising a portion of the amino acid sequence set forth in SEQ ID NO:2, wherein the amino acid sequence comprises at least one substitution set forth herein. In some further embodiments, the polynucleotide sequence encodes a polypeptide having an amino acid sequence that comprises at least one substitution at position 2, 3, 7, 11, 14, 17, 23, 24, 33, 36, 46, 47, 49, 56, 62, 68, 89, 97, 102, 108, 114, 116, 123, 132, 134, 143, 152, 155, 157, 162, 168, 184, 206, 219, 224, 225, 226, 228, 246, 231, 232, 233, 236, 240, 242, 245, 246, 249, 252, 255, 261, 266, 267, 275, 276, 279, 281, 282, 283, 285, 297, 301, 302, 303, and/or 318, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2. In some additional embodiments, the polynucleotide sequence encodes a polypeptide having an amino acid sequence that comprises at least one substitution selected from P2, S3, N7, D11, A14, F17, D23, V24, R33, K36, E46, D47, A49, A56, I62, K68, E89, S97, D102, L108, T114, K116, K123, K132, D134, I143, K152, K155, G157, I162, P168, S184, R206, Q219, L224, N225, Q226, R228, A246, N231, T232, S233, F236, T240, K242, A245, A246, G249, P252, V255, S261, A266, I267, P275, R276, E279, K281, D282, V283, S285, A297, I301, N302, L303, and/or V318, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2. In yet some additional embodiments, the polynucleotide sequence encodes a polypeptide having an amino acid sequence that comprises at least one substitution selected from P2T, S3H, S3R, S3W, N7L, D11K, A14V, F17W, D23E, V24G, R33L, R22V, K36Q, E46K, D47G, D47N, A49G, A56E, A56Y, 162V, K68G, K68M, K68R, E89N, E89V, S97R, S97T, D102T, L108Y, T114S, K116Q, K123C, K132A, K132N, D134E, D134H, D134V, I143L, K152A, K152E, K152H, K152Q, K155A, K155D, K155I, K155R, K155Y, G157R, I162L, P168S, S184A, R206S, R206V, Q219H, Q219L, Q219T, L224A, L224S, L224V, N225D, N225E, N225K, N225S, N225Y, Q226D, Q226E, Q226S, Q226V, R228T, N231G, N231H, N231L, N231S, T232A, T232C, T232S, T232V, S233C, S233F, S233G, S233I, S233K, S233V, F236L, T240V, K242L, A245S, A246L, A246S, G249D, P252C, V255I, S261A, S261C, S261N, S261T, A266V, A266C, 1267V, P275A, R276M, R276W, E279Q, K281L, K281V, D282C, D282G, D282R, V283H, S285E, S285T, A297H, A297S, I301C, I301Y, N302D, N302G, N302S, L303I, L303V, and/or V318C, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2. In some embodiments, the polynucleotide sequence is at least about 80%, about 85%, about 90%, or at least 95% identical to SEQ ID NO:1, and wherein the polynucleotide sequence comprises at least one mutation set forth herein. In some additional embodiments, the polynucleotide sequence comprises at least one mutation selected from t82a, t99a, g156a, c201t, a280c, c306t, t354g, t358c, a378t, c408t, a426t, c438t, a478c, t511c, a585g, t670c, t688c, t703c, t747c, t751a, t766c, t811a, c816t, a826c, c849g, c855t, and/or c906t, wherein the nucleotide position is determined by alignment with SEQ ID NO:1. In some additional embodiments, the present invention provides nucleic acid constructs comprising SEQ ID NO:3 and/or SEQ ID NO:4.

The present invention also provides isolated xylose reductase variants, wherein the variants have xylose reductase activity and comprise a substitution at one or more positions selected from 2, 3, 7, 11, 14, 17, 23, 24, 33, 36, 46, 47, 49, 56, 62, 68, 89, 97, 102, 108, 114, 116, 123, 132, 134, 143, 152, 155, 157, 162, 168, 184, 206, 219, 224, 225, 226, 228, 246, 231, 232, 233, 236, 240, 242, 245, 246, 249, 252, 255, 261, 266, 267, 275, 276, 279, 281, 282, 283, 285, 297, 301, 302, 303, and/or 318, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2. In some embodiments, the variant has xylose reductase activity and comprises a substitution at one or more positions selected from P2, S3, N7, D11, A14, F17, D23, V24, R33, K36, E46, D47, A49, A56, I62, K68, E89, S97, D102, L108, T114, K116, K123, K132, D134, I143, K152, K155, G157, I162, P168, S184, R206, Q219, L224, N225, Q226, R228, A246, N231, T232, S233, F236, T240, K242, A245, A246, G249, P252, V255, S261, A266, I267, P275, R276, E279, K281, D282, V283, S285, A297, I301, N302, L303, and/or V318, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2. In some additional embodiments, the variant has xylose reductase activity and comprises a substitution at one or more positions selected from P2T, S3H, S3R, S3W, N7L, D11K, A14V, F17W, D23E, V24G, R33L, R22V, K36Q, E46K, D47G, D47N, A49G, A56E, A56Y, 162V, K68G, K68M, K68R, E89N, E89V, S97R, S97T, D102T, L108Y, T114S, K116Q, K123C, K132A, K132N, D134E, D134H, D134V, I143L, K152A, K152E, K152H, K152Q, K155A, K155D, K155I, K155R, K155Y, G157R, I162L, P168S, S184A, R206S, R206V, Q219H, Q219L, Q219T, L224A, L224S, L224V, N225D, N225E, N225K, N225S, N225Y, Q226D, Q226E, Q226S, Q226V, R228T, N231G, N231H, N231L, N231S, T232A, T232C, T232S, T232V, S233C, S233F, S233G, S233I, S233K, S233V, F236L, T240V, K242L, A245S, A246L, A246S, G249D, P252C, V255I, S261A, S261C, S261N, S261T, A266V, A266C, 1267V, P275A, R276M, R276W, E279Q, K281L, K281V, D282C, D282G, D282R, V283H, S285E, S285T, A297H, A297S, I301C, I301Y, N302D, N302G, N302S, L303I, L303V, and/or V318C, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2. In some further embodiments, the isolated xylose reductase variants comprise the sequence set forth in SEQ ID NO:41, 43, 45, and/or 47. The present invention also provides isolated nucleic acid sequences comprising SEQ ID NO:3, 4, 40, 42, 44, and/or 46.

The present invention also provides recombinant nucleic acid constructs comprising at least one polynucleotide sequence that encodes a xylitol dehydrogenase, wherein the polynucleotide is selected from: (a) a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 70% identity to SEQ ID NO:6, wherein the amino acid sequence comprises at least one substitution set forth herein; and (b) a polynucleotide that hybridizes under stringent hybridization conditions to the complement of a polynucleotide that encodes a polypeptide having the amino acid sequence of SEQ ID NO:6, wherein the amino acid sequence comprises at least one substitution set forth herein. The present invention further provides nucleic acid constructs, wherein the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:6, wherein the amino acid sequence comprises at least one substitution set forth herein. In some embodiments, the polynucleotide sequence encodes a polypeptide comprising at least a portion of the amino acid sequence set forth in SEQ ID NO:6, wherein the amino acid sequence comprises at least one substitution set forth herein. In some further embodiments, the polynucleotide sequence encodes a polypeptide having an amino acid sequence that comprises at least one substitution at position 5, 13, 19, 49, 81, 149, 187, 189, 202, 205, 206, 210, 215, 218, 226, 227, 228, 229, 230, 231, 235, 239, 241, 251, 252, 256, 260, 286, 287, 296, 307, 327, 350, and/or 352, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some additional embodiments, the polynucleotide sequence encodes a polypeptide having an amino acid sequence that comprises at least one substitution selected from P5, D13, T19, A49, S81, H149, V187, L189, G202, V205, V206, I208, F209, D210, N211, M215, D218, F226, N227, S228, K229, T230, G231, E235, A239, G241, C251, T252, P256, L260, T286, V287, F296, K307, D327, A350, and/or K352, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some further embodiments, the polynucleotide sequence encodes a polypeptide having an amino acid sequence that comprises at least one substitution selected from P5E, P5R, D13G, T19L, T19Q, A49Q, S81G, H149R, V187M, L189C, G202D, V205C, V205H, V206A, I208R, F209S, D210W, N211K, N211R, N211S, M215A, M215C, D218R, F226V, N227T, S228P, K229R, T230V, G231A, G231H, E235K, E235Q, A239C, G241W, C251G, C251T, T252S, P256A, P256Q, L260Q, T286V, V287L, F296R, K307R, D327A, A350D, and/or K352E, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some still further embodiments, the polynucleotide sequence is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:5, and wherein the polynucleotide sequence comprises at least one mutation set forth herein. In some embodiments, the polynucleotide sequence comprises mutations at at least one position selected from t24g, c630t, a732g, a768t, a780g, wherein nucleotide position is determined by alignment with SEQ ID NO:5. In some additional embodiments, the nucleic acid construct comprises SEQ ID NO:7 and/or SEQ ID NO:8. In some further embodiments, the present invention provides an isolated nucleic acid sequence comprising SEQ ID NO:7, 8, and/or 48.

The present invention also provides isolated xylitol dehydrogenase variants comprising the sequence set forth in SEQ ID NO:49. In some additional embodiments, the present invention provides isolated xylitol dehydrogenase variants, wherein the variants have xylitol dehydrogenase activity and comprise a substitution at one or more positions selected from 5, 13, 19, 49, 81, 149, 187, 189, 202, 205, 206, 210, 215, 218, 226, 227, 228, 229, 230, 231, 235, 239, 241, 251, 252, 256, 260, 286, 287, 296, 307, 327, 350, and/or 352, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some embodiments, the xylitol dehydrogenase variant have xylitol dehydrogenase activity and comprise a substitution at one or more positions selected from P5, D13, T19, A49, S81, H149, V187, L189, G202, V205, V206, I208, F209, D210, N211, M215, D218, F226, N227, S228, K229, T230, G231, E235, A239, G241, C251, T252, P256, L260, T286, V287, F296, K307, D327, A350, and/or K352, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some further embodiments, the isolated xylitol dehydrogenase variant has xylitol dehydrogenase activity and comprises a substitution at one or more positions selected from P5E, P5R, D13G, T19L, T19Q, A49Q, S81G, H149R, V187M, L189C, G202D, V205C, V205H, V206A, I208R, F209S, D210W, N211K, N211R, N211S, M215A, M215C, D218R, F226V, N227T, S228P, K229R, T230V, G231A, G231H, E235K, E235Q, A239C, G241W, C251G, C251T, T252S, P256A, P256Q, L260Q, T286V, V287L, F296R, K307R, D327A, A350D, and/or K352E, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. The present invention also provides recombinant nucleic acid constructs comprising SEQ ID NO:11 and/or SEQ ID NO:12.

The present invention also provides recombinant nucleic acid constructs comprising polynucleotide sequences encoding at least one xylose reductase and at least one xylitol dehydrogenase, wherein the polynucleotide sequences are selected from SEQ ID NOS:1, 3, 4, 40, 42, 44, and 46; and SEQ ID NOS:5, 7, 8, 48.

The present invention further provides recombinant nucleic acid constructs further comprising at least one polynucleotide sequence encoding at least one xylulokinase, wherein the polynucleotide sequence comprises SEQ ID NO:11 and/or SEQ ID NO:12. In some embodiments, the recombinant nucleic acid constructs comprise at least one sequence selected from SEQ ID NOS:1, 3, 4, 5, 7, 8, 11, 12, 40, 42, 44, 46, and 48. In some further embodiments, the constructs comprise SEQ ID NO:46 and/or 48. In some additional embodiments, the nucleic acid constructs further comprise at least one genetic element that facilitates stable integration into a fungal host genome. In some additional embodiments, the nucleic acid constructs further comprise a genetic element that facilitates stable integration into a fungal host genome. In some embodiments, the genetic element facilitates integration into a fungal host genome by homologous recombination. In yet some additional embodiments, the nucleic acid constructs comprise a fungal origin of replication. In some embodiments, the fungal origin of replication is a yeast origin of replication. In some further embodiments, the polynucleotide sequence is operatively linked to a promoter sequence that is functional in a fungal cell. In some embodiments, the promoter sequence is a fungal promoter sequence. In some additional embodiments, the fungal promoter sequence is a yeast promoter sequence. In still some further embodiments, the polynucleotide sequence is operatively linked to a transcription termination sequence that is functional in a fungal cell. In some embodiments, the polynucleotide sequence contains codons optimized for expression in a yeast cell. In some further embodiments, the polynucleotide sequence comprises SEQ ID NO:3, 4, 7, 8, 11, and/or 12.

The present invention also provides recombinant fungal host cells comprising at least one polynucleotide sequence encoding at least one xylose reductase, wherein the polynucleotide sequences comprise at least one mutation relative to SEQ ID NO:1, as set forth herein. In some embodiments, the recombinant fungal host cell comprises at least one polynucleotide sequence selected from SEQ ID NOS:3, 4, 40, 42, 44, and 46. In some embodiments, the recombinant fungal host cells comprise at least one polynucleotide sequence encoding at least one xylitol dehydrogenase, wherein the polynucleotide sequence comprises at least one mutation relative to SEQ ID NO:5, as set forth herein. In some embodiments, at least one polynucleotide sequence is selected from SEQ ID NOS:7, 8, and 48.

The present invention also provides recombinant fungal host cells comprising at least one polynucleotide sequence encoding at least one xylulokinase, wherein the polynucleotide sequences comprise at least one mutation relative to SEQ ID NO:9. In some embodiments, at least one polynucleotide sequence is selected from SEQ ID NOS:11 and 12.

The present invention also provides recombinant fungal host cells comprising at least one polynucleotide sequence encoding at least one xylose reductase, wherein the polynucleotide sequence comprises SEQ ID NO:1, 3, 4, 40, 42, 44, and/or 46; and at least one polynucleotide encoding at least one xylitol dehydrogenase, wherein the polynucleotide sequence comprises SEQ ID NO:5, 7, 8, and/or 48. In some embodiments, the recombinant fungal host cells further comprise at least one polynucleotide encoding at least one xylulokinase, wherein the polynucleotide comprises SEQ ID NO:11 and/or 12. In some embodiments, at least one polynucleotide is integrated into the host cell genome. In some additional embodiments, the host cell is a yeast cell. In some embodiments, the host cell has had one or more native genes deleted from its genome. In some further embodiments, the deletion results in one or more phenotypes selected from increased transport of xylose into the host cell, increased xylulose kinase activity, increased flux through the pentose phosphate pathway, decreased sensitivity to catabolite repression, increased tolerance to ethanol, increased tolerance to acetate, increased tolerance to increased osmolarity, increased tolerance to low pH, and reduced production of by products, wherein comparison is made with respect to the corresponding host cell without the deletion(s). In some additional embodiments, the host cell is altered to overexpress one or more polynucleotides. In some further embodiments, overexpression results in one or more phenotypes selected from increased transport of xylose into the host cell, increased xylulose kinase activity, increased flux through the pentose phosphate pathway, decreased sensitivity to catabolite repression, increased tolerance to ethanol, increased tolerance to acetate, increased tolerance to increased osmolarity, increased tolerance to low pH, and reduced product of by products, wherein comparison is made to the corresponding unaltered host cell. In still some further embodiments, the host cell comprises at least one xylose reductase variant and at least one xylitol dehydrogenase variant. In some additional embodiments, the recombinant fungal host cells further comprise at least one xylulokinase. In some embodiments, the xylulokinase is encoded by SEQ ID NO:11 and/or 12. In some additional embodiments, the recombinant fungal host cell is transformed using nucleic acid constructs comprising from about two to about fifty copies of at least one xylose reductase and/or xylitol dehydrogenase and/or xylulokinase. It is not intended that the present invention be limited to any particular copy number of xylose reductase and/or xylitol dehydrogenase genes and/or xylulokinase, as any suitable number finds use in the present invention. In some embodiments, the nucleic acid constructs comprise any combination of wild-type and/or variant xylose reductase and/or xylitol dehydrogenase and/or xylulokinase. Furthermore, any suitable method for introducing multiple copies of either xylose reductase and/or xylitol dehydrogenase and/or xylulokinase find use in the present invention.

The present invention also provides processes for producing a fermentation product, comprising: (a) providing at least one recombinant fungal host cell provided herein; (b) providing a fermentation medium comprising; and (c) contacting the fermentation medium with the recombinant fungal host cell under conditions suitable for generating the fermentation product. In some embodiments, the process further comprises (d) recovering the fermentation product. In some additional embodiments, the fermenting step is carried out under conditions selected from microaerobic or aerobic conditions. In some embodiments, the fermenting step is carried out under anaerobic conditions. In some further embodiments, the fermentation product is selected from an alcohol, a fatty acid, lactic acid, acetic acid, 3-hydroxypropionic acid, acrylic acid, succinic acid, citric acid, malic acid, fumaric acid, succinic acid, an amino acid, 1,3-propanediol, ethylene, glycerol, and a β-lactam. In some additional embodiments, the fermentation product is an alcohol selected from ethanol and butanol. In some embodiments, the fermentation product is ethanol. In some embodiments, the fermentation medium comprises product from a saccharification process. In some further embodiments, the fermentation medium comprises hemicellulosic feedstock.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts xylose conversion pathways. In yeast and filamentous fungi, D-xylose is initially reduced to xylitol by NAD(P)H-dependent xylose reductase (“XR”). Xylitol is subsequently oxidized to D-xylulose by NAD+-dependent xylitol dehydrogenase (“XDH” or “XD”). Xylulokinase (“XK”) subsequently phosphorylates D-xylulose to produce D-xylulose 5-phosphate, which is then further metabolized through the pentose phosphate pathway (“PPP”). In bacteria, D-xylose is directly converted to D-xylulose by a xylose isomerase (“XI”).

FIGS. 2A-C depict the metabolic pathways for converting D-xylulose-5-P to ethanol.

FIG. 2A depicts the pentose phosphate pathway (PPP). The substrates and products are shown. The enzymes are represented by numbers as follows: 6. Ribulose-5-phosphate 3-epimerase; 7. Transketolase (TKL1); 8. Transaldolase (TAL1); 9. Ribose-5-phosphate ketoisomerase (RKI1); 10. 6-phosphogluconate dehydrogenase (GND1); 11. 6-phosphogluconalactonase (SOL3); and 12. Glucose-6-phosphate-1-dehydrogenase (ZWF).

FIG. 2B depicts the pathway of glycolysis. The substrates and products are shown. The enzymes are represented by numbers as follows: 13. Hexokinase; 14. Phosphoglucose isomerase; 15. Phosphofructokinase; 16. Aldolase; 17. Triose phosphate isomerase; 18. Glyceraldehyde 3-phosphate dehydrogenase; 19. 3-Phosphoglycerate kinase; 20. Phosphoglyceromutase; 21. Enolase; and 22. Pyruvate kinase.

FIG. 2C depicts the metabolic pathway for converting pyruvate to ethanol. The substrates and products are shown. The enzymes are represented by numbers as follows: 23. Pyruvate decarboxylase; 24. Aldehyde dehydrogenase; and 25. Alcohol dehydrogenase.

FIG. 3 provides a map of plasmid PLS2802, comprising XR.2, XD.2, and XK.2 genes.

DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions suitable for use in the conversion of xylose to xylitol and xylulose, including nucleic acid constructs, recombinant fungal host cells, and related materials.

All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference. Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, fermentation, microbiology, and related fields, which are known to those of skill in the art. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Indeed, it is intended that the present invention not be limited to the particular methodology, protocols, and reagents described herein, as these may vary, depending upon the context in which they are used. The headings provided herein are not limitations of the various aspects or embodiments of the present invention.

Nonetheless, in order to facilitate understanding of the present invention, a number of terms are defined below. Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

As used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

As used herein and in the appended claims, the singular “a”, “an” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “host cell” includes a plurality of such host cells.

Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. The headings provided herein are not limitations of the various aspects or embodiments of the invention that can be had by reference to the specification as a whole. Accordingly, the terms defined below are more fully defined by reference to the specification as a whole.

As used herein, the terms “isolated” and “purified” are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated.

As used herein, the term “reference enzyme” refers to an enzyme to which a variant enzyme of the present invention is compared in order to determine the presence of an improved property in the variant enzyme being evaluated, including but not limited to improved thermoactivity, improved thermostability, or improved stability. In some embodiments, a reference enzyme is a wild-type enzyme (e.g., wild-type xylose reductase or xylitol dehydrogenase). In some embodiments, a reference enzyme is another variant enzyme (e.g., another variant xylose reductase or xylitol dehydrogenase enzyme of the present invention).

As used herein, the term “recombinant” refers to a polynucleotide or polypeptide that does not naturally occur in a host cell. In some embodiments, recombinant molecules contain two or more naturally-occurring sequences that are linked together in a way that does not occur naturally. A recombinant cell contains a recombinant polynucleotide or polypeptide.

As used herein, the terms “enzyme variant” and “variant enzyme” are used in reference to enzymes that are similar to a reference enzyme, particularly in their function, but have mutations in their amino acid sequence that make them different in sequence from the wild-type or another reference enzyme. Enzyme variants (e.g., “xylose reductase variants” and/or “xylitol dehydrogenase variants”) can be made by a wide variety of different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available to make specific substitutions at defined amino acids (site-directed), specific or random mutations in a localized region of the gene (regio-specific) or random mutagenesis over the entire gene (e.g., saturation mutagensis). Numerous suitable methods are known to those in the art to generate enzyme variants, including but not limited to site-directed mutagenesis of single-stranded DNA or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. After the variants are produced, they can be screened for the desired property (e.g., high or increased; or low or reduced activity, increased thermal and/or alkaline stability, etc.).

As used herein, “combinatorial variant” refers to any variant that has a combination of two or more mutations (e.g., substitutions). In some embodiments, the combination of mutations results in changes in enzyme activity (e.g., improved thermostability, improved thermoactivity, improved specific activity, etc.).

As used herein, the term “overexpress” is intended to encompass increasing the expression of a protein to a level greater than the cell normally produces. It is intended that the term encompass overexpression of endogenous, as well as heterologous proteins.

For clarity, reference to a cell of a particular strain refers to a parental cell of the strain as well as progeny and genetically modified derivatives. Genetically modified derivatives of a parental cell include progeny cells that contain a modified genome or episomal plasmids that confer for example, antibiotic resistance, improved fermentation, the ability to utilize xylose as a carbon source, etc.

A nucleic acid construct, nucleic acid (e.g., a polynucleotide), polypeptide, or host cell is referred to herein as “recombinant” when it is non-naturally occurring, artificial or engineered.

The terms “xylose reductase” and “xylose reductase polypeptide” are used interchangeably herein to refer to an enzyme that is capable of catalyzing xylose to xylitol. The ability to catalyze xylose to xylitol is referred to herein as “xylose reductase activity”.

The terms “protein” and “polypeptide” are used interchangeably herein to refer to a polymer of amino acid residues. The term “xylose reductase polynucleotide” refers to a polynucleotide that encodes a xylose reductase polypeptide.

In some embodiments, xylose reductase polynucleotides employed in the practice of the present invention encode a polypeptide comprising an amino acid sequence that is at least about 71% identical, at least about 72% identical, at least about 73% identical, at least about 74% identical, at least about 75% identical, at least about 76% identical, at least about 77% identical, at least about 78% identical, at least about 79% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO:2, wherein the sequence comprises at least one substitution set forth herein. In some embodiments, the xylose reductase polynucleotide encodes a polypeptide having an amino acid sequence comprising SEQ ID NO:41, 43, 45, and/or 47.

In some embodiments, xylose reductase polynucleotides employed in the practice of the present invention comprise polynucleotide sequences that are at least about 70% identical, at least about 71% identical, at least about 72% identical, at least about 73% identical, at least about 74% identical, at least about 75% identical, at least about 76% identical, at least about 77% identical, at least about 78% identical, at least about 79% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at last about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO:1, 3 and/or 4.

The terms “xylitol dehydrogenase” and “xylitol dehydrogenase polypeptide” are used interchangeably herein to refer to an enzyme that is capable of catalyzing xylitol to xylulose. The ability to catalyze xylitol to xylulose is referred to herein as “xylitol dehydrogenase activity”. Also, as used herein, the term “xylitol dehydrogenase polynucleotide” refers to a polynucleotide that encodes a xylitol dehydrogenase polypeptide.

In some embodiments, xylitol dehydrogenase polynucleotides employed in the practice of the present invention encode polypeptides comprising amino acid sequences that are at least about 71% identical, at least about 72% identical, at least about 73% identical, at least about 74% identical, at least about 75% identical, at least about 76% identical, at least about 77% identical, at least about 78% identical, at least about 79% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO:6, wherein SEQ ID NO:6 further comprises at least one substitution as set forth herein. In some embodiments, the xylitol dehydrogenase polynucleotide encodes a polypeptide having an amino acid sequence comprising SEQ ID NO:6 and/or 49, wherein SEQ ID NO:6 further comprises at least one substitution as set forth herein

In some embodiments, xylulokinase polynucleotides employed in the practice of the present invention comprise polynucleotides sequence that are at least about 70% identical, at least about 71% identical, at least about 72% identical, at least about 73% identical, at least about 74% identical, at least about 75% identical, at least about 76% identical, at least about 77% identical, at least about 78% identical, at least about 79% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at last about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO:9, 11 and/or 12, wherein SEQ ID NO:9 further comprises at least one substitution.

The terms “percent identity,” “% identity”, “percent identical,” and “% identical,” are used interchangeably herein to refer to the percent amino acid or polynucleotide sequence identity that is obtained by ClustalW analysis (version W 1.8 available from European Bioinformatics Institute, Cambridge, UK), counting the number of identical matches in the alignment and dividing such number of identical matches by the length of the reference sequence, and using the following ClustalW parameters to achieve slow/accurate pairwise optimal alignments—DNA/Protein Gap Open Penalty: 15/10; DNA/Protein Gap Extension Penalty: 6.66/0.1; Protein weight matrix: Gonnet series; DNA weight matrix: Identity; Toggle Slow/Fast pairwise alignments=SLOW or FULL Alignment; DNA/Protein Number of K-tuple matches: 2/1; DNA/Protein number of best diagonals: 4/5; DNA/Protein Window size: 4/5.

Two sequences are “aligned” when they are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences. Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well known in the art (See, e.g., Dayhoff et al., in Dayhoff [ed.], Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3, Natl. Biomed. Res. Round., Washington D.C. [1978]; pp. 345-352; and Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919 [1992], both of which are incorporated herein by reference). The BLOSUM62 matrix is often used as a default scoring substitution matrix in sequence alignment protocols such as Gapped BLAST 2.0. The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The alignment is defined by the amino acid position of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences so as to arrive at the highest possible score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer-implemented alignment algorithm (e.g., gapped BLAST 2.0; See, Altschul et al., Nucleic Acids Res., 25:3389-3402 [1997], which is incorporated herein by reference), and made available to the public at the National Center for Biotechnology Information Website). Optimal alignments, including multiple alignments can be prepared using readily available programs such as PSI-BLAST (See e.g., Altschul et al., supra).

The present invention also provides a recombinant nucleic acid construct comprising a xylose reductase polynucleotide sequence that hybridizes under stringent hybridization conditions to the complement of a polynucleotide which encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, wherein the polypeptide is capable of catalyzing the xylose to xylitol, and the polypeptide comprises at least one substitution set forth herein.

In some embodiments, the polynucleotide that hybridizes to the complement of a polynucleotide which encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, wherein the polypeptide comprises at least one substitution as set forth herein, does so under high or very high stringency conditions to the complement of a reference sequence having the sequence of SEQ ID NO:1 (e.g., over substantially the entire length of the reference sequence). In some embodiments, the polynucleotide that hybridizes to the complement of a polynucleotide which encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, wherein the polypeptide comprises at least one substitution as set forth herein, does so under high or very high stringency conditions to the complement of a reference sequence having the sequence of SEQ ID NO:3 (e.g., over substantially the entire length of the reference sequence). In some embodiments, the polynucleotide that hybridizes to the complement of a polynucleotide which encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, wherein the amino acid sequence comprises at least one substitution as set forth herein, does so under high or very high stringency conditions to the complement of a reference sequence having the sequence of SEQ ID NO:4 (e.g., over substantially the entire length of the reference sequence).

The present invention also provides a recombinant nucleic acid construct comprising a xylitol dehydrogenase polynucleotide sequence that hybridizes under stringent hybridization conditions to the complement of a polynucleotide which encodes a polypeptide having the amino acid sequence of SEQ ID NO:6, wherein the polypeptide comprises at least one substitution set forth herein, and wherein the polypeptide is capable of catalyzing xylitol to xylulose.

Nucleic acids “hybridize” when they associate, typically in solution. There are numerous texts and other reference materials that provide details regarding hybridization methods for nucleic acids (See e.g., Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes,” Part 1, Chapter 2, Elsevier, New York, [1993], which is incorporated herein by reference). For polynucleotides of at least 100 nucleotides in length, low to very high stringency conditions are defined as follows: prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures. For polynucleotides of at least 200 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at least at 50° C. (low stringency), at least at 55° C. (medium stringency), at least at 60° C. (medium-high stringency), at least at 65° C. (high stringency), and at least at 70° C. (very high stringency).

The terms “corresponding to”, “reference to” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

The “position” is denoted by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus. Owing to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminal will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where there is a deletion in an aligned test sequence, there will be no amino acid that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to any amino acid position the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

As used herein, the terms “transformed” and “transformation” used in reference to a cell refer to a cell that has a non-native nucleic acid sequence integrated into its genome or has an episomal plasmid that is maintained through multiple generations.

As used herein, the term “by-product” refers to an organic molecule that is an undesired product of a particular fermentation process.

As used herein, the term “xylose pathway” refers to the steps of conversion of xylose to xylulose phosphate which is then metabolized through the pentose phosphate pathway. In some embodiments, this involves the reduction of xylose to xylitol, oxidation of xylitol to xylulose and subsequent conversion of xylulose to xylulose phosphate. In some other embodiments the xylose is directly converted to xylulose which is then phosphorylated to xylulose phosphate.

As used herein the term “xylose pathway enzymes” refers to the enzymes that catalyze the conversion of xylose to xylulose-phosphate. In some embodiments, these enzymes comprise xylose reductase, xylitol dehydrogenase and/or xylulose kinase. In some other embodiments, the enzymes comprise xylose isomerase and xylulose kinase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions suitable for use in the conversion of xylose to xylitol and xylulose, including nucleic acid constructs, recombinant fungal host cells, and related materials.

The initial metabolic pathways for xylose utilization in fungi and bacteria differ. In most fungi, including xylose-fermenting yeasts (e.g., Pichia stipitis, Pachysolen tannophilus, and Candida shehatae), D-xylose is converted to D-xylulose by two oxidoreductases involving cofactors NAD(P)H and NAD(P)+. (See, Matsushika et al., Appl. Microbiol. Biotechnol., 84:37-53 [2009]). In these organisms, D-xylose is initially reduced to xylitol by NAD(P)H-dependent xylose reductase (“XR”) (EC 1.1.1.21). Xylitol is subsequently oxidized to D-xylulose by NAD+-dependent xylitol dehydrogenase (“XDH” or “XD”) (EC 1.1.1.9). Xylulokinase (“XK”) (EC 2.7.1.17) subsequently phosphorylates D-xylulose to produce D-xylulose 5-phosphate (“X5P”), which is then further metabolized through the pentose phosphate pathway (“PPP”). FIG. 1 provides a schematic of the pathway.

However, most strains of S. cerevisiae cannot utilize xylose even though the genes encoding XR, XDH, and XK are present in its genome, as the expression levels of these enzymes are too low to allow xylose utilization (See, Matsushika et al., supra). Some strains have been shown to natively utilize xylose but at very low rates and fermentation to ethanol has not been detected (See, Wenger et al., PLoS Genet., 6(5):e1000942 [2010]). Even when the endogenous genes are overexpressed in S. cerevisiae, only slow growth on xylose has been observed (See, Matsushika et al. supra).

In contrast, most bacteria (e.g., Escherichia coli and Streptomyces species) can isomerize D-xylose directly to D-xylulose by using a xylose isomerase (“XI”) (EC 5.3.1.5) (See, Matsushika et al., supra). In bacteria, as in fungi, the D-xylulose is phosphorylated to D-xylulose 5-phosphate by XK, which is then further metabolized through the pentose phosphate pathway.

Xylose utilization by these host cells results in useful products that are produced metabolically by the host cell. In these host cells, D-xylulose may be phosphorylated by a native or recombinant xylulokinase to xylulose-5-P, as depicted in FIG. 1. The xylulose-5-P may be further metabolized by enzymes in the pentose phosphate pathway to products such as glucose-6-P, fructose-6-P, glyceraldehydes-3-P, and the like. The pentose phosphate pathway and relevant enzymes and products are depicted in FIG. 2A. As used herein, the terms “enzyme from the pentose phosphate pathway” and “pentose phosphate pathway enzyme” are used interchangeably to refer to an enzyme from the group of enzymes involved in the pentose phosphate pathway, (i.e., 6. ribulose-5-phosphate ketoisomerase (RK11); 7. transketolase (TKL1); 8. transaldolase (TAL1); 9. ribose-5-phosphate ketolisomerase (RK11); 10. 6-phosphogluconate dehydrogenase (GND1); 11. 6-phosphogluconalactonase (SOL3); and/or 12. glucose-6-phosphate-1-dehydrogenase (ZWF); the reference numbers correspond to those in FIG. 2A).

Products of the pentose phosphate pathway may be further metabolized through the process of glycolysis. The metabolic process of glycolysis is depicted in FIG. 2B. As used herein, the term “glycolytic enzyme” refers to an enzyme from the group of enzymes involved in glycolysis (i.e.: 13. hexokinase; 14. phosphoglucose isomerase; 15. phosphofructokinase; 16. aldolase; 17. triose phosphate isomerase; 18. glyceraldehyde phosphate dehydrogenase; 19. phosphoglycerate kinase; 20. phosphglyceromutase; 21. enoase; and/or 22. pyruvate kinase; the reference numbers correspond to those in FIG. 2B).

Pyruvate from the glycolytic pathway (i.e., glycolysis) may be further metabolized to ethanol as shown in FIG. 2C by ethanologenic enzymes. As used herein, the term “ethanologenic enzyme” refers to an enzyme involved in the conversion of pyruvate to ethanol, (e.g., a pyruvate decarboxylase, an aldehyde dehydrogenase, and/or an alcohol dehydrogenase). The term “ethanologenic pathway” refers to the pathway depicted in FIG. 2C.

Recombinant host cells transformed with xylose reductase and xylitol dehydrogenase genes are hence capable of converting xylose to xylitol and then converting xylitol to xylulose, which can lead to the production of desirable fermentation products (e.g., an alcohol, such as ethanol, butanol, and the like, including, but not limited to a fatty alcohol [e.g., a C8-C20 fatty alcohol], a fatty acid [e.g., a C8-C20 fatty acid], lactic acid, 3-hydroxpropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propanediol, ethylene, glycerol, a β-lactam, and the like). However, cells transformed with wildtype xylose reductase and xylitol dehydrogenase genes from Pichia stipitis convert xylose inefficiently and with accumulation of xylitol (Matsushika et al., Appl. Environ Microbiol., 81:243-55 [2008]). The present application provides improved xylose reductase and xylitol dehydrogenase variants that significantly increase the efficiency of xylose conversion.

Recombinant Nucleic Acid Constructs

The present invention provides recombinant nucleic acid constructs comprising polynucleotide sequences that encode at least one polypeptide comprising an amino acid sequence having at least 70% identity to SEQ ID NO:2, wherein the polypeptide is capable of catalyzing xylose to xylitol, and wherein the polypeptide comprises at least one substitution set forth herein. SEQ ID NO:2 corresponds to the amino acid sequence encoding xylose reductase from the yeast, Pichia stipitis. SEQ ID NO:1 corresponds to the native P. stipitis polynucleotide sequence that encodes a P. stipitis xylose reductase (SEQ ID NO:2), while SEQ ID NOS:3 and 4 correspond to codon-optimized sequences encoding the xylose reductase. In some embodiments, the nucleic acid construct comprises SEQ ID NO:40, while in other embodiments, the nucleic acid construct comprises SEQ ID NO:42, in still other embodiments, the nucleic acid construct comprises SEQ ID NO:44, and in additional embodiments, the nucleic acid construct comprises SEQ ID NO:46. In some embodiments, the polypeptide comprises SEQ ID NO:41, while in other embodiments, the polypeptide comprises SEQ ID NO:43, in still other embodiments, the polypeptide comprises SEQ ID NO:45, and in further embodiments, the polypeptide comprises SEQ ID NO:47.

The present invention also provides a recombinant nucleic acid constructs comprising polynucleotide sequences that encode at least one polypeptide comprising an amino acid sequence having at least 70% identity to SEQ ID NO:6, wherein the polypeptide is capable of catalyzing xylitol to xylulose, and wherein the polypeptide comprises at least one substitution set forth herein. SEQ ID NO:6 corresponds to the amino acid sequence encoding xylitol dehydrogenase from the yeast, Pichia SEQ ID NO:5 corresponds to the native P. stipitis polynucleotide sequence that encodes a P. stipitis xylitol dehydrogenase (SEQ ID NO:6), while SEQ ID NOS:7 and 8 correspond to codon-optimized sequences encoding the xylitol dehydrogenase. In some embodiments, the nucleic acid construct comprises SEQ ID NO:48, while in other embodiments, the nucleic acid construct encodes a polypeptide comprising SEQ ID NO:49.

The present invention also provides recombinant nucleic acid constructs comprising polynucleotide sequences that encode polypeptide sequences having xylose reductase and xylitol dehydrogenase activity. In some embodiments, the nucleic acid constructs comprise at least one of SEQ ID NO:40, 42, 44, and/or 46, and SEQ ID NO:48.

The present invention also provides nucleic acid constructs comprising codon-optimized polynucleotides encoding xylulokinase. In some embodiments, the present invention provides SEQ ID NO:11, while in other embodiments, the present invention provides SEQ ID NO:12.

The present invention also provides nucleic acid constructs comprising polynucleotides encoding xylose reductase, xylitol dehydrogenase and xylulokinase. In some embodiments, the nucleic acid constructs comprise SEQ ID NO:1, 3, 4, 40, 42, 44, and/or 46, as well as SEQ ID NO: 5, 7, 8, and/or 48, and SEQ ID NO:9, 11, and/or 12.

In some embodiments, recombinant nucleic acid constructs of the present invention further comprise a polynucleotide sequence (genetic) element that facilitates integration into a fungal host cell genome, by homologous or non-homologous recombination. In some embodiments, the nucleic acid construct of the present invention further comprises an origin of replication that is functional in a fungal cell (e.g., a yeast origin of replication). Typically, the fungal host cell is a yeast or filamentous fungal cell, more typically, a yeast cell. In some embodiments, nucleic acid constructs of the present invention comprise a transcriptional regulatory element that is functional in a fungal cell. For example, in some embodiments the recombinant nucleic acid construct comprises a promoter sequence and/or transcription terminator sequence that is functional in a fungal cell such that the xylose reductase and/or xylitol dehydrogenase polynucleotide is operatively linked to the promoter sequence and/or transcription terminator sequences.

Xylose reductase and xylitol dehydrogenase polynucleotides that are suitable for use in the practice of the present invention include those encoding variants of SEQ ID NO:2 and/or 6, respectively. These variants include those having amino acid sequences with one or more conservative or non-conservative substitutions relative to the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO:6. As used herein, the term “conservative substitution” refers to the substitution of a residue for another residue that does not generally alter the specific activity of the encoded polypeptide. An exemplary conservative substitution is a substitution that is within the same group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine, proline, cysteine and methionine). Amino acid substitutions that do not generally alter the specific activity are well-known in the art. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr. Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly, as well as these in reverse.

In some embodiments, polynucleotides encoding conservatively substituted variations of the P. stipitis xylose reductase and/or xylitol dehydrogenase employed in the practice of the present invention include substitutions of a small percentage, typically less than about 5%, more typically less than 2%, and often less than about 1% of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group.

Other xylose reductase and/or xylitol dehydrogenase polynucleotides suitable for use in the practice of the present invention include those encoding variants of P. stipitis xylose reductase and/or xylitol dehydrogenase generated by mutagenesis, recombination, or other protein engineering method followed by screening of the variants for xylose utilization using a method, such as that described in the Examples. In some embodiments, the resulting variants comprise one or more substitutions (conservative or non-conservative), deletions, and/or insertions. The present invention thus provides methods for making improved P. stipitis xylose reductase and xylitol dehydrogenase polynucleotide variants, wherein the method comprises introducing one or more modifications into a polynucleotide encoding SEQ ID NO:1, 3 and/or 4; and/or 5, 7 and/or 8, respectively, to produce a modified polynucleotide, wherein the modification is selected from at least one substitution, at least one deletion, and/or at least one insertion; transforming a host cell with the modified polynucleotide; and screening the transformed host cell for an improvement in a desired phenotype relative to the corresponding untransformed host cell. In some embodiments, the improved variants are screened to assess improvements in a desired phenotype relative to a transformed host cell that has been transformed with wild-type sequences, while in other embodiments, transformed host cells are compared with other transformed host cells. Exemplary phenotypes include improved utilization of a pentose sugar (e.g., xylose, arabinose, etc.), stability, specific activity, lower Ki for xylitol, ethanol/acetate tolerance and/or tolerance to low pH, decreased by-product formation, and/or increased ethanol yield. Exemplary desirable xylose utilization phenotypes include the ability to ferment xylose to ethanol, the ability to ferment xylose to other metabolic intermediates/products, the ability to undergo aerobic or anaerobic growth on xylose, and the like.

Methods for generating variant libraries of polynucleotides encoding modified polypeptides are well known in the art. For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides encoding the xylose reductase polypeptide of SEQ ID NO:2 and/or xylitol dehydrogenase of SEQ ID NO:6, to generate variant libraries that can be expressed, screened, and assayed using the methods described herein. Mutagenesis and directed evolution methods are well known in the art (See e.g., Ling et al., Anal. Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of which are incorporated herein by reference).

In some embodiments, the present invention provides P. stipitis polypeptide variants that comprise at least one modification that is a substitution, insertion, and/or deletion relative to SEQ ID NO:2. Typically, the polypeptide variant has from 1 to 2, 1 to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, up to about 50, 75, 100, or 130 modifications.

In some embodiments, the xylose reductase variants of the present invention comprise a substitution at position 276 of wild-type P. stipitis xylose reductase. In some alternative embodiments, the xylose reductase variants comprise at least one substitution at positions 2, 49, 132, 233, 267, and/or 276, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2. In some additional embodiments, the substitutions comprise substitutions at R276, P2, A49, K132, S233, I267, and/or R276. In some further embodiments, the substitutions comprise R276W, P2T, A49G, K132N, S233K, I267V, and/or R276W, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2. In some embodiments, the variants comprise substitutions P2T, A49G, and R276W, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2. In some additional embodiments, the variants comprise substitutions P2T, A49G, K132N, S233K, I267V, and R276W, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2. In some additional embodiments, the variants comprise at least one substitution selected from position 2, 3, 7, 11, 14, 17, 23, 24, 33, 36, 46, 47, 49, 56, 62, 68, 89, 97, 102, 108, 114, 116, 123, 132, 134, 143, 152, 155, 157, 162, 168, 184, 206, 219, 224, 225, 226, 228, 246, 231, 232, 233, 236, 240, 242, 245, 246, 249, 252, 255, 261, 266, 267, 275, 276, 279, 281, 282, 283, 285, 297, 301, 302, 303, and 318, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2.

In still some further embodiments, the variants comprise at least one substitution selected from P2, S3, N7, D11, A14, F17, D23, V24, R33, K36, E46, D47, A49, A56, I62, K68, E89, S97, D102, L108, T114, K116, K123, K132, D134, I143, K152, K155, G157, I162, P168, S184, R206, Q219, L224, N225, Q226, R228, A246, N231, T232, S233, F236, T240, K242, A245, A246, G249, P252, V255, S261, A266, I267, P275, R276, E279, K281, D282, V283, S285, A297, I301, N302, L303, and V318, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2.

In some additional embodiments, the substitutions comprise at least one substitution selected from P2T, S3H, S3R, S3W, N7L, D11K, A14V, F17W, D23E, V24G, R33L, R22V, K36Q, E46K, D47G, D47N, A49G, A56E, A56Y, 162V, K68G, K68M, K68R, E89N, E89V, S97R, S97T, D102T, L108Y, T114S, K116Q, K123C, K132A, K132N, D134E, D134H, D134V, I143L, K152A, K152E, K152H, K152Q, K155A, K155D, K155I, K155R, K155Y, G157R, I162L, P168S, S184A, R206S, R206V, Q219H, Q219L, Q219T, L224A, L224S, L224V, N225D, N225E, N225K, N225S, N225Y, Q226D, Q226E, Q226S, Q226V, R228T, N231G, N231H, N231L, N231S, T232A, T232C, T232S, T232V, S233C, S233F, S233G, S233I, S233K, S233V, F236L, T240V, K242L, A245S, A246L, A246S, G249D, P252C, V255I, S261A, S261C, S261N, S261T, A266V, A266C, I267V, P275A, R276M, R276W, E279Q, K281L, K281V, D282C, D282G, D282R, V283H, S285E, S285T, A297H, A297S, I301C, I301Y, N302D, N302G, N302S, L303I, L303V, and V318C, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:2.

In some embodiments, the present invention provides polynucleotide sequences encoding xylose reductase variants comprising SEQ ID NO:40, as provided below. In some additional embodiments, the present invention provides polypeptide sequences comprising SEQ ID NO:41, as provided below.

(SEQ ID NO: 40) ATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTT TCGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTACCG TGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGCC AACGAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTA TCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTA CCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGAC TTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCT TCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGG TAAGGGTGACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGG AAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGGTCTATCGGTG TTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTAC CATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAA CCAAGATTGATCGAGTTCGCTCAATCCCGTGGTATTGCTGTCACCGCTT ACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGC TTTGAACACTTCTCCATTGTTCGAGAACGAAACTATCAAGGCTATCGCT GCTAAGCACGGTAAGTCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCC AAAGAGGCATTGCCATCATTCCAAAGTCCAACACTGTCCCATGGTTGTT GGAAAACAAGGATGTCAACAGCTTCGACTTGGACGAACAAGATTTCGCT GACATTGCCAAGTTGGACATCAACTTGAGATTCAACGACCCATGGGACT GGGACAAGATTCCTATCTTCGTCTAA (SEQ ID NO: 41) MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDYA NEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSD LQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETW KALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQ PRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIA AKHGKSPAQVLLRWSSQRGIAIIPKSNTVPWLLENKDVNSFDLDEQDFA DIAKLDINLRFNDPWDWDKIPIFV

In some embodiments, the present invention provides polynucleotide sequences encoding xylose reductase variants comprising SEQ ID NO:42, as provided below. In some additional embodiments, the present invention provides polypeptide sequences comprising SEQ ID NO:43, as provided below.

(SEQ ID NO: 42) ATGACCTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGT TTCGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTAC CGTGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTAC GGCAACGAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAA GGTATCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAAC AACTACCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTT TCTGACTTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCA GTCACCTTCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTC TACTGTGGTAAGGGTGACAACTTCGACTACGAAGATGTTCCAATTTTA GAGACCTGGAAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGG TCTATCGGTGTTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTG AGAGGTGCTACCATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCA TACTTGCAACAACCAAGATTGATCGAGTTCGCTCAATCCCGTGGTATT GCTGTCACCGCTTACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTG AACCAAGGTAGAGCTTTGAACACTTCTCCATTGTTCGAGAACGAAACT ATCAAGGCTATCGCTGCTAAGCACGGTAAGTCTCCAGCTCAAGTCTTG TTGAGATGGTCTTCCCAAAGAGGCATTGCCATCATTCCAAAGTCCAAC ACTGTCCCATGGTTGTTGGAAAACAAGGATGTCAACAGCTTCGACTTG GACGAACAAGATTTCGCTGACATTGCCAAGTTGGACATCAACTTGAGA TTCAACGACCCATGGGACTGGGACAAGATTCCTATCTTCGTCTAA (SEQ ID NO: 43) MTSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDY GNEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTL SDLQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPIL ETWKALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHP YLQQPRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENET IKAIAAKHGKSPAQVLLRWSSQRGIAIIPKSNTVPWLLENKDVNSFDL DEQDFADIAKLDINLRFNDPWDWDKIPIFV

In some embodiments, the present invention provides polynucleotide sequences encoding xylose reductase variants comprising SEQ ID NO:44, as provided below. In some additional embodiments, the present invention provides polypeptide sequences comprising SEQ ID NO:45, as provided below.

(SEQ ID NO: 44) ATGACCTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGT TTCGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTAC CGAGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTAC GGCAACGAAAAATTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAA GGTATCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAAC AACTACCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTT TCTGACTTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCA GTCACCTTCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTC TACTGTGGTAACGGTGACAACTTCGACTACGAAGATGTTCCAATTTTA GAGACCTGGAAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGG TCTATCGGTGTTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTG AGAGGTGCTACCATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCA TACTTGCAACAACCAAGATTGATCGAGTTCGCTCAATCCCGTGGTATT GCTGTCACCGCTTACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTG AACCAAGGTAGAGCTTTGAACACTAAGCCATTGTTCGAGAACGAAACT ATCAAGGCTATCGCTGCTAAGCACGGCAAGAGCCCAGCTCAAGTCTTG TTGAGATGGTCTTCCCAAAGAGGCATTGCCGTTATTCCAAAGTCCAAC ACTGTCCCATGGTTGTTGGAAAACAAGGATGTCAACAGCTTCGACTTG GACGAACAAGATTTCGCTGACATTGCCAAGTTGGACATCAACTTGAGA TTCAACGACCCATGGGACTGGGACAAGATTCCTATCTTCGTCTAA (SEQ ID NO: 45) MTSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDY GNEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTL SDLQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGNGDNFDYEDVPIL ETWKALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHP YLQQPRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTKPLFENET IKAIAAKHGKSPAQVLLRWSSQRGIAVIPKSNTVPWLLENKDVNSFDL DEQDFADIAKLDINLRFNDPWDWDKIPIFV

In some embodiments, the present invention provides polynucleotide sequences encoding xylose reductase variants comprising SEQ ID NO:46, as provided below. In some additional embodiments, the present invention provides polypeptide sequences comprising SEQ ID NO:47, as provided below.

(SEQ ID NO: 46) ATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGT TTCGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTAC CGTGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTAC GCCAACGAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAA GGTATCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAAC AACTACCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTT TCTGACTTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCA GTCACCTTCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTC TACTGTGGTAAGGGTGACAACTTCGACTACGAAGATGTTCCAATTTTA GAGACCTGGAAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGG TCTATCGGTGTTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTG AGAGGTGCTACCATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCA TACTTGCAACAACCAAGATTGATCGAGTTCGCTCAATCCCGTGGTATT GCTGTCACCGCTTACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTG AACCAAGGTAGAGCTTTGAACACTTCTCCATTGTTCGAGAACGAAACT ATCAAGGCTATCGCTGCTAAGCACGGTAAGTCTCCAGCTCAAGTCTTG TTGAGATGGTCTTCCCAAAGAGGCATTGCCATCATTCCAAAGAGCAAT ACTGTCCCATTCTTGTTGGAAAACAAGGATGTCAACAGCTTCGACTTG GACGAACAAGATTTCGCTGACATTGCCAAGTTGGACATCAACTTGAGA TTCAACGACCCATGGGACTGGGACAAGATTCCTATCTTCGTCTAA (SEQ ID NO: 47) MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDY ANEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTL SDLQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPIL ETWKALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHP YLQQPRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENET IKAIAAKHGKSPAQVLLRWSSQRGIAIIPKSNTVPFLLENKDVNSFDL DEQDFADIAKLDINLRFNDPWDWDKIPIFV

In some embodiments, the present invention also provides nucleic acid substitutions in sequences encoding xylose reductase variants (e.g., SEQ ID NOS:1, 3 and/or 4). In some embodiments, the nucleic acids comprise substitutions at positions 42, 82, 99, 156, 201, 280, 306, 354, 358, 378, 408, 438, 478, 511, 585, 670, 688, 703, 747, 751, 766, 855, 849, and/or 906, wherein the positions are numbered by correspondence with the nucleic acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:1, 3 and/or 4. In some embodiments, the substitutions comprise t82a, t99a, g156a, c201t, a280c, c306t, t354g, t358c, a378t, c408t, a426t, c438t, a478c, t511c, a585g, t670c, t688c, t703c, t747c, t751a, t766c, c849g, c855t, and/or c906t, wherein the positions are numbered by correspondence with the nucleic acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:1, 3 and/or 4.

In some embodiments, the present invention further provides xylitol dehydrogenase variants comprising at least one substitution at positions 5, 13, 19, 49, 81, 149, 187, 189, 202, 205, 206, 208, 209, 211, 215, 218, 226, 227, 228, 229, 231, 235, 239, 241, 251, 252, 256, 260, 287, 296, 307, 327, 350, and/or 352, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:6.

In some embodiments, the xylitol dehydrogenase variants comprise substitution at position 208, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some embodiments, the substitution at position 208 is I208X. In some further embodiments, the substitution at position 208 is I208R. In some embodiments, the xylitol dehydrogenase variants comprise substitutions at position 211, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some embodiments, the substitution at position 211 is N211X. In some further embodiments, the substitution is N211K, while in other embodiments, the substitution is N211S, and in still other embodiments, the substitution is N211K.

In some embodiments, the xylitol dehydrogenase variants comprise substitutions at positions 208 and 211, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some embodiments, the substitution at position 208 is I208X and the substitution at position 211 is N211X. In some further embodiments, the substitutions comprise I208R and N211K, while in some other embodiments, the substitutions comprise I208R and N211S.

In some embodiments, the xylitol dehydrogenase variants comprise substitutions at positions 209 and 211, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some embodiments, the substitutions are F209X and N211X.

In some embodiments, the xylitol dehydrogenase variants comprise substitutions at positions 208, 209 and 211, wherein the positions are numbered by correspondence with the amino acid sequence of P. stipitis xylitol dehydrogenase set forth in SEQ ID NO:6. In some embodiments, the substitutions are I208X, F209X, and N211X. In some further embodiments, the substitutions are I208R, F209S, and N211S.

In some embodiments, the present invention also provides nucleic acid substitutions in sequences encoding xylitol dehydrogenase variants (e.g., SEQ ID NOS:5, 7, and/or 8). In some embodiments, the nucleic acids comprise substitutions at positions t24g, c630t, a732g, a768t, and/or a780g, wherein the positions are numbered by correspondence with the nucleic acid sequence of P. stipitis xylose reductase set forth in SEQ ID NO:5, 7 and/or 8.

In some embodiments, the present invention provides polypeptides that comprise XR and XD substitutions, including, but not limited to combinations of substitutions at positions 271, 270, 272, and/or 276 in XR (wherein the positions are numbered by correspondence with SEQ ID NO:2) and substitutions at positions 208, 209, and/or 211 in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6). In some embodiments, the substitutions comprise substitutions S271X, K270, N272, and/or R276 in XR (wherein the positions are numbered by correspondence with SEQ ID NO:2) and substitutions I208X, F209X, and/or N211X in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6). In some embodiments, the substitutions comprise substitutions S271G, K270R, N272P, and/or R276F in XR (wherein the positions are numbered by correspondence with SEQ ID NO:2) and substitutions I208R, F209S, and/or N211K in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6).

In some embodiments, the substitutions comprise R276F in XR (wherein the position is numbered by correspondence with SEQ ID NO:2), and I208R in XD (wherein the position is numbered by correspondence with SEQ ID NO:6). In some embodiments, the substitutions comprises R276F in XR (wherein the position is numbered by correspondence with SEQ ID NO:2), and F209S and N211K in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6). In some embodiments, the substitutions comprise S271G in XR (wherein the position is numbered by correspondence with SEQ ID NO:2), and I208R and N211K in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6). In some embodiments, the substitutions comprise S271G in XR (wherein the position is numbered by correspondence with SEQ ID NO:2), and I208R and N211S in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6). In some embodiments, the substitutions comprise R276F in XR (wherein the position is numbered by correspondence with SEQ ID NO:2), and I208R, F209S and N211K in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6). In some embodiments, the substitutions comprise S271G in XR (wherein the position is numbered by correspondence with SEQ ID NO:2), and I208R in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6). In some embodiments, the substitutions comprise K270R in XR (wherein the position is numbered by correspondence with SEQ ID NO:2), and I208R and N211R in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6). In some embodiments, the substitutions comprise K270R in XR (wherein the position is numbered by correspondence with SEQ ID NO:2), and F209S and N211K in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6). In some embodiments, the substitutions comprise N272P and R276F in XR (wherein the position is numbered by correspondence with SEQ ID NO:2), and F209S and N211K in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6). In some embodiments, the substitutions comprise K270R, N272P and R276F in XR (wherein the position is numbered by correspondence with SEQ ID NO:2), and N211K in XD (wherein the positions are numbered by correspondence with SEQ ID NO:6).

In some additional embodiments, the present invention provides XR and XD variants having amino acid and nucleic acid substitutions, wherein amino acid substitutions in SEQ ID NO:2 comprises R276F, amino acid substitutions in SEQ ID NO:6 comprise I208R, and the nucleic acid substitutions comprise t811a and c816t in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the present invention provides XR and XD variants having amino acid and nucleic acid substitutions, wherein amino acid substitutions in SEQ ID NO:2 comprise R276F, amino acid substitutions in SEQ ID NO:6 comprise F209S and N211K, and the nucleic acid substitutions comprise t811a and c816t in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the present invention provides XR and XD variants having amino acid and nucleic acid substitutions, wherein amino acid substitutions in SEQ ID NO:2 comprise S271G, amino acid substitutions in SEQ ID NO:6 comprise I208R and N211K, and the nucleic acid substitutions comprise c816t and a826c in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the present invention provides XR and XD variants having amino acid and nucleic acid substitutions, wherein amino acid substitutions in SEQ ID NO:2 comprise S271G, amino acid substitutions in SEQ ID NO:6 comprise I208R and N211S, and the nucleic acid substitutions comprise c816t and a826c in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the present invention provides XR and XD variants having amino acid and nucleic acid substitutions, wherein amino acid substitutions in SEQ ID NO:2 comprise R276F, amino acid substitutions in SEQ ID NO:6 comprise I208R, F209S and N211K, and the nucleic acid substitutions comprise t811a and c816t in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the present invention provides XR and XD variants having amino acid and nucleic acid substitutions, wherein amino acid substitutions in SEQ ID NO:2 comprise S271G, amino acid substitutions in SEQ ID NO:6 comprise I208R, and the nucleic acid substitutions comprise c816t and a826c in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the present invention provides XR and XD variants having amino acid and nucleic acid substitutions, wherein amino acid substitutions in SEQ ID NO:2 comprise K270R, amino acid substitutions in SEQ ID NO:6 comprise I208R and N211R, and the nucleic acid substitutions comprise t811a, c816t, and a826c, in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the present invention provides XR and XD variants having amino acid and nucleic acid substitutions, wherein amino acid substitutions in SEQ ID NO:2 comprise K270R, amino acid substitutions in SEQ ID NO:6 comprise F209S and N211K, and the nucleic acid substitutions comprise t811a, c816t, and a826c, in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the present invention provides XR and XD variants having amino acid and nucleic acid substitutions, wherein amino acid substitutions in SEQ ID NO:2 comprise K272P and R276F, amino acid substitutions in SEQ ID NO:6 comprise F209S and N211K, and the nucleic acid substitutions comprise t811a in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the present invention provides XR and XD variants having amino acid and nucleic acid substitutions, wherein amino acid substitutions in SEQ ID NO:2 comprise K270R, N272P, and R276F, amino acid substitutions in SEQ ID NO:6 comprise N211K, and the nucleic acid substitutions comprise t811a in SEQ ID NO:1, 3 and/or 4. In some additional embodiments, the present invention provides XR and XD variants having amino acid and nucleic acid substitutions, wherein amino acid substitutions in SEQ ID NO:6 comprise I208R, and the nucleic acid substitutions comprise t811a, c816t, and a826c, in SEQ ID NO:1, 3 and/or 4.

In some embodiments, the present invention provides polynucleotide sequences encoding xylitol dehydrogenase variants comprising SEQ ID NO:48, as provided below. In some additional embodiments, the present invention provides polypeptide sequences comprising SEQ ID NO:49, as provided below.

(SEQ ID NO: 48) ATGACCGCTAATCCCTCTCTTGTTTTGAATAAGATTGACGACATTTCT TTTGAAACTTACGATGCTCCCGAAATTAGCGAACCCACAGACGTTTTA GTTCAAGTTAAAAAAACTGGTATCTGCGGTTCTGACATCCACTTCTAC GCTCATGGAAGGATCGGCAACTTCGTCTTAACAAAGCCAATGGTTCTG GGTCATGAAAGCGCGGGTACTGTTGTTCAAGTCGGTAAAGGTGTTACT TCACTGAAGGTTGGTGATAACGTCGCAATCGAGCCCGGTATTCCATCT AGGTTCAGTGATGAGTACAAATCTGGTCACTACAACCTGTGTCCACAC ATGGCATTTGCTGCTACTCCCAATTCTAAAGAGGGTGAACCAAACCCA CCAGGAACTCTATGTAAGTACTTCAAATCTCCAGAAGACTTCCTGGTT AAGTTACCCGATCATGTTTCTTTGGAGTTGGGTGCTTTGGTCGAGCCA CTATCTGTTGGGGTCCATGCTAGTAAATTAGGCTCCGTTGCATTTGGC GATTACGTTGCTGTTTTTGGTGCTGGTCCAGTAGGATTACTGGCTGCC GCTGTCGCTAAGACATTTGGTGCCAAGGGTGTGATTGTCGTTGATATA TCTGACAAGAAGCTGAAGATGGCCAAAGACATAGGTGCCGCTACACAT ACCTTCAACTCCAAGACGGGAGGTAGTGAAGAATTGATCAAAGCCTTC GGTGGTAATGTACCAAATGTTGTCTTGGAATGTACTGGGGCTGAACCA TGTATTAAGCTAGGTGTTGATGCCATCGCACCAGGTGGTAGATTCGTG CAAGTTGGTAATGCTGCTGGTCCCGTGTCCTTTCCCATAACAGTGTTC GCTATGAAAGAACTTACTTTGTTTGGTTCATTTCGTTATGGTTTCAAC GACTATAAGACAGCCGTGGGTATCTTTGATACTAACTACCAGAACGGT AGAGAGAATGCTCCCATTGACTTTGAACAGCTTATCACGCACAGATAC AAATTCAAAGACGCCATTGAAGCCTACGACCTAGTAAGAGCAGGTAAA GGGGCTGTCAAGTGTTTGATTGATGGTCCAGAATAA (SEQ ID NO: 49) MTANPSLVLNKIDDISFETYDAPEISEPTDVLVQVKKTGICGSDIHFY AHGRIGNFVLTKPMVLGHESAGTVVQVGKGVTSLKVGDNVAIEPGIPS RFSDEYKSGHYNLCPHMAFAATPNSKEGEPNPPGTLCKYFKSPEDFLV KLPDHVSLELGALVEPLSVGVHASKLGSVAFGDYVAVFGAGPVGLLAA AVAKTFGAKGVIVVDISDKKLKMAKDIGAATHTFNSKTGGSEELIKAF GGNVPNVVLECTGAEPCIKLGVDAIAPGGRFVQVGNAAGPVSFPITVF AMKELTLFGSFRYGFNDYKTAVGIFDTNYQNGRENAPIDFEQLITHRY KFKDAIEAYDLVRAGKGAVKCLIDGPE

In some embodiments, the present invention provides polynucleotide sequences encoding xylose reductase variants comprising SEQ ID NO:46 and xylitol dehydrogenase variants comprising SEQ ID NO:48. In some additional embodiments, the present invention provides polypeptide sequences comprising SEQ ID NO:47 and SEQ ID NO:49.

Also suitable for use in the practice of the present invention are polynucleotides encoding a truncated variant of P. stipitis xylose reductase and/or a truncated variant xylitol dehydrogenase capable of catalyzing xylose to xylitol and/or xylitol to xylulose. In some embodiments, these truncation variants are truncated at the carboxy (C)-terminus and/or the amino (N)-terminus. Typically, the truncation is from about 1 to about 50 amino acid residues in length.

Those having ordinary skill in the art will understand that due to the degeneracy of the genetic code, a multitude of nucleotide sequences that encode the xylose isomerase polypeptides described herein exist. Table 1 provides the standard triplet genetic code for each amino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in the nucleic acids referred to herein, where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence. The invention contemplates and provides each and every possible variation of nucleic acid sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices.

TABLE 1 Genetic Code Amino Acids Codon Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

In some embodiments, DNA sequences are designed for high codon usage bias (i.e., codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid). In some embodiments, the preferred codons are determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. In particular, a DNA sequence can be optimized for expression in a particular host organism. References providing preference information for a wide range of organisms are readily available (See e.g., Henaut and Danchin in Neidhardt et al. [eds.], Escherichia coli and Salmonella, ASM Press, Washington D.C., [1987], p. 2047-2066, which is incorporated herein by reference).

A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (See, GCG CodonPreference, Genetics Computer Group Wisconsin Package; Peden, Codon W, University of Nottingham; McInerney, Bioinform., 14:372-73 [1998]; Stenico et al., Nucl. Acids Res. 222437-46 [1994]; Wright, Gene 87:23-29 [1990]; Wada et al., Nucl. Acids Res., 20:2111-2118 [1992]; Nakamura et al., Nucl. Acids Res., 28:292 [2000]; and Henaut and Danchin, supra; all of which are incorporated herein by reference). The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to express proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTs), or predicted coding regions of genomic sequences (See e.g., Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., [2001]; Uberbacher, Methods Enzymol., 266:259-281 [1996]; and Tiwari et al., Comput. Appl. Biosci. 13:263-270 [1997]; all of which are incorporated herein by reference).

In some embodiments, the xylose reductase and xylitol dehydrogenase polynucleotides contain codons optimized for expression in a fungal cell, particularly a yeast cell. In some embodiments, codon-optimized xylose reductase polynucleotide sequence is provided as SEQ ID NOS:3 and 4. In addition, codon-optimized xylitol dehydrogenase polynucleotides are provided herein (SEQ ID NOS:7 and 8), as well as codon-optimized xylulokinase polynucleotides (SEQ ID NOS:11 and 12). In some embodiments, the codon-optimized sequences provide various advantages, as compared to the corresponding wild-type S. cerevisiae sequence(s).

Certain silent mutations have been identified in P. stipitis xylose reductase and xylitol dehydrogenase polynucleotide variants that appear to confer the property of greater xylose utilization in transformed Saccharomyces cerevisiae. These variants are described in the Examples. The silent mutations include t24g, t82a, t99a, g156a, c201t, a280c, c306t, t354g, t358c, a378t, c408t, a426t, c438t, a478c, t511c, a585g, c630t, t670c, t688c, t703c, a732g, t747c, t751a, t766c, a768t, a780g, t811a, c816t, a826c, c849g, c855t, and c906t (where the nucleotide position is determined by alignment with SEQ ID NO:1). However, it is not intended that the present invention be limited to these particular substitutions as alternative substitutions find use in the present invention.

In some embodiments, the xylose reductase and/or xylitol dehydrogenase polynucleotides are employed in recombinant nucleic acid constructs that comprise a vector (e.g., a plasmid, a cosmid, a phage, a virus, a yeast artificial chromosome (YAC), and the like), into which a xylose reductase and/or xylitol dehydrogenase polynucleotide sequence has been inserted. The xylose reductase and xylitol dehydrogenase polynucleotides provided herein find use when incorporated into any one of a variety of vectors. Suitable vectors include, but are not limited to chromosomal, nonchromosomal and synthetic DNA sequences, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and many others. Any suitable vector that transduces genetic material into a cell, and, if replication is desired, which is replicable and viable in the relevant host find use in the present invention.

Nucleic acid constructs of the present invention find use in transforming a host cell to permit the host to express the xylose reductase and/or xylitol dehydrogenase polypeptide. Methods for recombinant expression of proteins in fungi are well known in the art, and a number of vectors are available or can be constructed using routine methods (See e.g., Zhu et al., Plasmid 6:128-33 [2009], incorporated herein by reference; and the many standard reference works in this field).

In some embodiments, recombinant nucleic acid constructs of the present invention further comprise a transcriptional regulatory element that is functional in a fungal cell. In some embodiments, the nucleic acid construct comprises the xylose reductase and/or xylitol dehydrogenase polynucleotide operatively linked to a transcriptional regulatory sequence (e.g., a promoter, transcription termination sequence, and the like), that is functional in a fungal cell. Examples of promoters that are functional in a fungal host cell include, but are not limited to promoters from yeast and filamentous fungi. Promoters that are suitable for use in the practice of the present invention include endogenous or heterologous promoters and include both constitutive and inducible promoters that are natural or modified. Particularly useful promoters are those that are insensitive to catabolite (glucose) repression and/or do not require xylose for induction. Such promoters are well known in the art. In some embodiments, a promoter sequence is operably linked to the 5′ region of the xylose isomerase or xylitol dehydrogenase coding sequence using routine methods that are well known in the art.

Promoters that are suitable for use in the practice of the present invention include, but are not limited to yeast promoters from glycolytic genes (e.g., yeast phosphofructokinase (PFK), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase (PGK) promoters, and the like; See e.g., WO 93/03159, which is incorporated herein by reference); promoters of glucose transporters; ribosomal protein encoding gene promoters; alcohol dehydrogenase promoters (e.g., ADH1, ADH4, and the like), and the enolase promoter (ENO).

Exemplary promoters that are useful for directing the transcription of the nucleic acid constructs of the present invention in yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae enolase (eno-1), Saccharomyces cerevisiae galactokinase (gal1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1/ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae transcription elongation factor (TEF), Saccharomyces cerevisiae fructose 1,6-bisphosphate aldolase (FBA1), and Saccharomyces cerevisiae 3-phosphate glycerate kinase (PGK1). Other useful promoters for yeast host cells are well known in the art (See e.g., Romanos et al., Yeast 8:423-488 [1992], which is incorporated herein by reference).

Suitable filamentous fungal promoters that are useful in the practice of the present invention include, but are not limited to promoters obtained from the genes for Aspergillus oryzeae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96/00787, which is incorporated herein by reference), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae those phosphate isomerase), promoters such as cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2, xyn1, amy, and glaA (See, Nunberg et al., Mol. Cell Biol., 4:2306-2315 [1984]; Boel et al., EMBO J. 3:1581-85 [1984]; and EP 0 137 280A, all of which are incorporated herein by reference), and mutant, truncated, and hybrid promoters thereof. Promoters associated with chitinase production in fungi also find use in some embodiments (See e.g., Blaiseau and Lafay, Gene 120:243-248 [1992] [filamentous fungus Aphanocladium album]; and Limon et al., Curr. Genet, 28:478-83 [1995] [Trichoderma harzianum]; both of which are incorporated herein by reference).

Any other suitable promoter sequence that drives expression in a fungal host cell, particularly a yeast host cell finds use in the present invention. Suitable promoter sequences can be identified using well known methods. In one approach, a putative promoter sequence is linked 5′ to a sequence encoding a reporter protein, the construct is transfected into the host cell and the level of expression of the reporter is measured. Expression of the reporter can be determined by measuring, for example, mRNA levels of the reporter sequence, an enzymatic activity of the reporter protein, or the amount of reporter protein produced. For example, promoter activity may be determined by using the green fluorescent protein as coding sequence (See, Henriksen et al., Microbiol., 145:729-34 [1999], which is incorporated herein by reference) or a lacZ reporter gene (See, Punt et al., Gene, 197:189-93 [1997], which is incorporated herein by reference). In some embodiments, functional promoters are derived from naturally occurring promoter sequences by directed evolution methods (See e.g., Wright et al., Hum. Gene Ther., 16:881-892 [2005], which is incorporated herein by reference).

Exemplary transcription termination sequences (terminators) that are functional in a fungal host cell, include transcription termination sequences from yeast and filamentous fungi, that are well known in the art. In some embodiments, the transcription termination sequence is from a yeast. Exemplary yeast transcription termination sequences include, but are not limited to CYC1, ADH1t, ADH2t, etc. In some embodiments, the nucleic acid constructs of the present invention contain a ribosome binding site for translation initiation. In some embodiments, the construct includes appropriate sequences for amplifying expression (e.g., an enhancer). Such elements are well known in the art and any suitable enhancers and/or transcription termination sequences, and/or ribosome binding sites find use in the present invention.

In some additional embodiments, nucleic acid constructs of the present invention contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Suitable marker genes include, but are not limited to those coding for antimicrobial resistance such as, ampicillin (ampR), kanamycin, chloramphenicol, tetracycline, streptomycin or spectinomycin (e.g., the aada gene); including but not limited to the streptomycin phosphotransferase (spt) gene coding for streptomycin resistance, the neomycin phosphotransferase (nptII) gene encoding kanamycin or geneticin resistance, the nourseothricin actetyltransferase (nat1) gene coding for nourseothricin resistance, the hygromycin phosphotransferase (hpt) gene coding for hygromycin resistance, genes encoding dihydrofolate reductase, phleomycin, or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance in E. coli, as well as other marker genes that are well known in the art.

Nucleic acid constructs of the present invention typically comprise a fungal origin of replication, such as, for example, a filamentous fungal or yeast origin of replication. Typically, the recombinant nucleic acid constructs of the present invention comprise a yeast origin of replication. Examples include, but are not limited to constructs containing autonomous replicating sequences, constructs containing 2 micron DNA including the autonomous replicating sequence and rep genes, constructs containing centromeres like the CEN6, CEN4, CEN11, CDN3 and autonomous replicating sequences, and other like sequences that are well known in the art. Exemplary nucleic acid constructs include constructs suitable for transforming yeast. These include, but are not limited to episomal constructs based on the yeast 2μ or CEN origin based plasmids like pYES2/CT, pYES3/CT, pESC/His, pESC/Ura, pESC/Trp, pES/Leu, p427TEF, pRS405, pRS406, pRS413, and other yeast-based constructs that are known in the art.

In some embodiments, the nucleic acid constructs of the present invention comprise elements to facilitate integration of the xylose reductase and/or xylitol dehydrogenase polynucleotide into a fungal host chromosome (i.e., the genome), by either homologous or non-homologous recombination and either site-directed or random mutagenesis. In some embodiments, the nucleic acid constructs comprise elements that facilitate homologous integration. In some embodiments, the xylose reductase and/or xylitol dehydrogenase polynucleotide is integrated at one or more site and is present in one or more copies. In some embodiments, the nucleic acid construct comprises the xylose reductase and/or xylitol dehydrogenase polynucleotide(s) and no promoter that is operatively linked to the xylose reductase and/or xylitol dehydrogenase polynucleotide. This type of construct typically comprises genetic elements to facilitate integration into the fungal host chromosome at a location that is downstream of a native promoter (i.e., in the host chromosome). In some embodiments, a second nucleic acid construct is employed which comprises a promoter and genetic elements to facilitate integration into the fungal host chromosome in a location upstream of the targeted integration site of the xylose reductase and/or xylitol dehydrogenase polynucleotide. In some embodiments, the nucleic acid construct comprises the xylose reductase and/or xylitol dehydrogenase polynucleotide operatively linked to a promoter or promoter and terminator sequences such that all are integrated into the host chromosome (genome).

Genetic elements that facilitate integration by homologous recombination are those having sequence homology to targeted integration sites in the fungal host chromosome (genome). Suitable sites that find use as targets for integration include, but are not limited to the TY1 loci, the RDN loci, the ura3 locus, the GPD locus, aldose reductase (GRE3) locus, etc. Those having ordinary skill in the art appreciate that additional sites for integration can be readily identified using methods known in the art, including but not limited to microarray analysis, metabolic flux analysis, comparative genome hybridization analysis, etc.

Genetic elements or techniques which facilitate integration by non-homologous recombination include, but are not limited to restriction enzyme-mediated integration (REMI) (See e.g., Manivasakam et al., Mol. Cell Biol., 18(3):1736-1745 [1998], which is incorporated herein by reference), transposon-mediated integration, and other elements and methods that are well known in the art.

In some embodiments, the nucleic acid constructs of the present invention comprise at least one further recombinant polynucleotide that is capable of conferring a desired phenotype to a fungal host cell, particularly in the context of xylose fermentation. In some embodiments, the recombinant polynucleotide that is capable of conferring an improved phenotype to the fungal host cell is a non-coding polynucleotide such as a regulatory polynucleotide, a coding polynucleotide, or combination thereof.

Exemplary further desired phenotypes include, but are not limited to increased transport of xylose into the host cell, increased xylulose kinase activity, increased flux through the pentose phosphate pathway, decreased sensitivity to catabolite repression, increased tolerance to ethanol, increased tolerance to increased osmolarity, increased tolerance to organic acids, reduced production of by-products, and other similar properties related to increasing flux through the pentose phosphate and glycolysis pathways to produce a desired metabolic product/intermediate at higher levels as compared to the corresponding wild-type host cell. Typically, the desired metabolic product is an alcohol (e.g., ethanol).

In some embodiments, nucleic acid constructs comprising at least one further polynucleotide that is capable of conferring a desired phenotype to a fungal host cell comprise a polynucleotide encoding a protein known to impact the desired phenotype, wherein the polynucleotide is either native or heterologous to the fungal host cell. In some embodiments, this polynucleotide is operatively linked to its native promoter, or to a heterologous promoter (i.e., a promoter that is not associated with the polynucleotide in the corresponding native gene). In some embodiments, the at least one further polynucleotide is overexpressed. In some embodiments, the nucleic acid constructs comprise multiple copies of a least one polynucleotide. Suitable polynucleotides include, but are not limited to those that facilitate overexpression of proteins known to have an impact on the desired phenotype.

Exemplary recombinant polynucleotides that are capable of conferring a desired phenotype to a fungal host cell include recombinant polynucleotides (either wild-type or mutated forms) which encode a xylose or hexose transporter, a xylulose kinase (XKS), an enzyme from the pentose phosphate pathway (See e.g., FIG. 2A), a glycolytic enzyme (i.e., from the glycolytic metabolic pathway; See e.g., FIG. 2B), and an ethanologenic enzyme (See e.g., FIG. 2C), regulatory sequences that enhance expression of these sequences, and combinations thereof. Additional recombinant polynucleotides (either wild-type or mutated forms) that find use in the present invention include those that encode additional proteins involved in the pentose phosphate, glycolysis, and ethanologenic pathways (See e.g., FIGS. 2A-C).

Exemplary transporters include, but are not limited to GXF1, SUT1 and At6g59250 from Candida intermedia, Pichia stipitis and Arabidopsis thaliana, respectively (See e.g., Runquist et al., Biotechnol. Biofuels., 3:5 [2010], which is incorporated herein by reference), as well as HXT4, HXT5, HXT7, GAL2, AGT1, GXF2 (See e.g., Matsushika et al., Appl. Microbiol. Biotechnol., 84:37-53 [2009], which is incorporated herein by reference). In some embodiments, overexpression of native S. cerevisiae transporters is desirable, particularly HXT5 and HXT7.

Particularly suitable recombinant polynucleotides include those which encode: a xylulose kinase (XK); an enzyme from the pentose phosphate pathway (e.g., a ribulose-5-phosphate 3-epimerase (RPE1), a ribose-5-phosphate ketol-isomerase (RKI1), a transketolase (TKL1), a transaldolase (TAL1), etc.); a glycolytic enzyme (e.g., a hexokinase (HXK1/HXK2), a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pyruvate kinase (PVK2), etc.); and an ethanologenic enzyme (e.g., a pyruvate decarboxylase, an alcohol dehydrogenase, etc.).

Exemplary regulatory polynucleotides include promoters, enhancer, terminator, and other regulatory elements that function to improve the expression of polynucleotides in a fungal host cell, particularly, a yeast host cell. These include, but are not limited to the regulatory elements described hereinabove.

The nucleic acid constructs described herein are useful for transforming fungal host cells to confer to these cells the property of xylose utilization.

Recombinant Fungal Host Cells

The present invention provides recombinant fungal host cells comprising at least one xylose reductase and/or xylitol dehydrogenase polynucleotide provided herein. More specifically, the recombinant fungal host cell comprises a polynucleotide sequence that encodes a polypeptide which is capable of catalyzing xylose to xylitol, wherein the polynucleotide is selected from: (a) a polynucleotide that encodes a polypeptide comprising an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein the polypeptide comprises at least one substitution set forth herein; and (b) a polynucleotide that hybridizes under stringent hybridization conditions to the complement of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO:2, wherein the polypeptide comprises at least one substitution set forth herein. In some other embodiments, the recombinant fungal host cell comprises a polynucleotide sequence that encodes a polypeptide which is capable of catalyzing xylitol to xylulose, wherein the polynucleotide is selected from: (a) a polynucleotide that encodes a polypeptide comprising an amino acid sequence that is at least about 70% identical to SEQ ID NO:6, wherein the polypeptide comprises at least one substitution as set forth herein; and (b) a polynucleotide that hybridizes under stringent hybridization conditions to the complement of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO:6, wherein the polypeptide comprises at least one substitution set forth herein. In some embodiments, the recombinant fungal host cell comprises polynucleotide sequences that encode a polypeptide which is capable of catalyzing xylose to xylitol and a polypeptide capable of catalyzing xylitol to xylulose-5-P, wherein the polynucleotides are selected from: (a) polynucleotides that encode polypeptides comprising amino acid sequences that are at least about 70% identical to SEQ ID NO:2 and/or 6, wherein each of the polypeptides comprise at least one substitution set forth herein; and (b) polynucleotides that hybridize under stringent hybridization conditions to the complement of polynucleotides encoding polypeptides having the amino acid sequences of SEQ ID NO:2 and/or 6, wherein each of the polypeptides comprise at least one substitution set forth herein.

In some embodiments, the recombinant fungal host cell further comprises at least one xylulokinase, including but not limited to the xylulokinase provided herein (SEQ ID NO:10), encoded by SEQ ID NO:9, and by codon-optimized sequences SEQ ID NOS:11 and 12.

In some embodiments, the recombinant fungal host cell comprises at least one xylose reductase, at least one xylitol dehydrogenase, and at least one xylulokinase. In some embodiments, the xylose reductase comprises SEQ ID NO:2, 41, 43, 45, and/or 47, In some embodiments, the xylitol dehydrogenase comprises SEQ ID NO:6 and/or 42. In some embodiments, the xylulokinase comprises SEQ ID NO:10.

In some embodiments, the present invention provides a recombinant fungal host cell comprising or transformed with a nucleic acid construct of the present invention. In some embodiments, the xylose reductase and/or xylitol dehydrogenase and/or xylulokinase polynucleotide is integrated into the host cell genome. Typically, the recombinant fungal host cell is a filamentous fungal or yeast host cell. More typically, the recombinant fungal host cell is a yeast host cell.

The present invention also provides methods for producing a recombinant fungal host cell, wherein the method comprises: (a) providing at least one nucleic acid construct of the present invention, wherein the nucleic acid construct comprises at least one xylose reductase and/or xylitol dehydrogenase and/or at least one xylulokinase polynucleotide provided herein; and (b) transforming a fungal host cell with the nucleic acid construct to produce a recombinant fungal host cell. In some additional embodiments, the recombinant fungal host cell is transformed using nucleic acid constructs comprising from about two to about fifty copies of at least one xylose reductase and/or xylitol dehydrogenase and/or xylulokinase. It is not intended that the present invention be limited to any particular copy number of xylose reductase and/or xylitol dehydrogenase genes and/or xylulokinase, as any suitable number of copies finds use in the present invention. In some embodiments, the nucleic acid constructs comprise any combination of wild-type and/or variant xylose reductase and/or xylitol dehydrogenase and/or xylulokinase. Furthermore, any suitable method for introducing multiple copies of either xylose reductase and/or xylitol dehydrogenase and/or xylulokinase find use in the present invention.

Introduction of the expression construct of the present invention into the host cell can be accomplished using any suitable method, including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation, or any other suitable technique. Indeed, there are numerous methods known in the art and described in various standard reference texts. In some embodiments, the xylose reductase and/or xylitol dehydrogenase and/or xylulokinase polynucleotide sequence is integrated into the host cell genome.

Suitable fungal host cells include yeast and filamentous fungal host cells. In some embodiments, the fungal host cell is a yeast cell. Exemplary yeast host cells that are useful in the practice of the present invention include, but are not limited to Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, and Yarrowia. In some embodiments of the invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica. In some embodiments, the yeast host cell is Saccharomyces species. In some additional embodiments, the yeast host cell is Saccharomyces cerevisiae.

Any suitable yeast strain finds use in the present invention, including but not limited to strains such as those commercially available from companies such as Lallemand (e.g., Superstart™, THERMOSACC®, etc.); RED STAR and ETHANOL RED® (Fermentis/Lesaffre); FALI (AB Maui); (Baker's Best Yeast, Baker's Compressed Yeast, etc. (Fleishmann's Yeast); BIOFERM AFT, XP, CF, and XR (North American Bioproducts Corp.); Turbo Yeast (Gert Strand AB); FERMIOL® (DSM Specialties); BY4741 (e.g., ATCC 201388); Y108-1 (ATCC PTA.10567) and LNH-ST (See, US Pat. Publ. No. 2011/0159560); NRRL YB-1952 (ARS Culture Collection), as well as any additional suitable strains.

In some embodiments, the filamentous fungal host cell is a species of Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothia, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, Volvariella, or teleomorphs, or anamorphs, and synonyms or taxonomic equivalents thereof. However, it is not intended that the present invention be limited to any particular species of filamentous fungal host cell. Exemplary filamentous fungal host cells that find use in the present invention include, but are not limited to a filamentous fungal host cell of the Trichoderma species (e.g., T. longibrachiatum, T. viride [e.g., ATCC 32098 and 32086], T. reesei [NRRL 15709, ATTC 13631, 56764, 56765, 56466, 56767, and RL-P37 and derivatives thereof; See e.g., Sheir-Neiss et al., Appl. Microbiol. Biotechnol., 20:46-53 [1984], incorporated herein by reference), T. koningii, and T. harzianum), as well as Hypocrea jecorina. The term “Trichoderma” refers to any fungal strain that was previously classified as Trichoderma or is currently classified as Trichoderma.

In some embodiments of the present invention, the filamentous fungal host cell is an Aspergillus species (e.g., A. awamori, A. funigatus, A. japonicas, A. nidulans, A. niger, A. aculeatus, A. foetidus, A. oryzae, A. sojae, or A. kawachi (See e.g., Kelly and Hynes, EMBO J., 4:475479 [1985]; NRRL 3112, ATCC 11490, 22342, 44733, and 14331; Yelton et al., Proc. Natl. Acad. Sci. USA, 81, 1480-1474 [1984]; Tilburn et al., Gene 26, 205-221 [1982]; and Johnston et al., EMBO J., 4:1307-1311 [1985], all of which are incorporated herein by reference). In some embodiments of the invention, the filamentous fungal host cell is a Fusarium species (e.g., F. bactridioides, F. cerealis, F. crookwellense, F. culmorum, F. graminaearum, F. graminum, F. oxysporum, F. rosium, or F. venenatum). In some embodiments of the invention, the filamentous fungal host cell is of a Neurospora species (e.g., N. crassa; See e.g., Case, et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]; U.S. Pat. No. 4,486,553; and Kinsey and Rambosek, Mol. Cell. Biol., 4:117-122 [1984], all of which are incorporated herein by reference). In some embodiments of the invention, the filamentous fungal host cell is of a Humicola species (e.g., H. insolens, H. grisea, or H. lanuginose). In some embodiments of the invention, the filamentous fungal host cell is a Mucor species (e.g., M. miehei or M. circinelloides). In some embodiments of the invention, the filamentous fungal host cell is a Rhizopus species (e.g., R. oryzae or R. niveus). In some embodiments of the invention, the filamentous fungal host cell is of a Penicillum species (e.g., P. purpurogenum, P. chrysogenum, or P. verruculosum). In some embodiments of the invention, the filamentous fungal host cell is a Thielavia species (e.g., T. terrestris). In some embodiments of the invention, the filamentous fungal host cell is a Tolypocladium species (e.g., T. inflatum or T. geodes). In some embodiments of the invention, the filamentous fungal host cell is a Trametes species (e.g., T. villosa or T. versicolor). In some embodiments of the invention, the filamentous fungal host cell is a Chrysosporium species, (e.g., C. lucknowense, C. keratinophilum, C. tropicum, C. merdarium, C. inops, C. pannicola, or C. zonatum).

Strains that find use in the present invention include those that are readily accessible to the public from a number of culture collection, including but not limited to the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkutlturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

Recombinant fungal host cells of the present invention are capable of fermenting xylose when provided with a xylose based culture medium. Typically the recombinant fungal host cells described herein are capable of fermenting xylose at a faster rate compared to the corresponding wild-type fungal host cell. In some embodiments, the recombinant fungal host cells provided herein are capable of fermenting xylose at a faster rate compared to fungal host cells transformed with a nucleic acid construct comprising xylose reductase and/or xylitol dehydrogenase genes. In some other embodiments, the recombinant fungal host cells provided herein are capable of fermenting xylose at a faster rate compared to fungal host cells transformed with a nucleic acid construct comprising of xylose reductase, xylitol dehydrogenase and/or xylulose kinase genes. In some embodiments, the recombinant fungal host cells are capable of fermenting xylose at a rate of at least about 1 g/L/h and sometimes at a rate of at least about 2 g/L/h. Exemplary xylose-based culture media include culture media which have been formulated to contain xylose, as well as feedstock from cellulosic saccharification processes and/or feedstock from a hemicellulose pre-treatment process (i.e., a “hemicellulosic feedstock”).

In some embodiments, the fungal host cell is a wild-type fungal cell, while in other embodiments, it is a mutated or otherwise altered or engineered form of a wild-type fungal cell. Typically, the fungal host cell (either wild-type or otherwise altered or engineered) comprises polynucleotides encoding a xylulokinase and one or more enzymes in the pentose phosphate, glycolytic, and/or ethanologenic pathways. In some embodiments, the fungal host cell comprises polynucleotides encoding a xylulokinase and all of the enzymes in the pentose phosphate, glycolytic, and ethanologenic pathways. In some embodiments, the fungal host cell comprises recombinant polynucleotides encoding enzymes that are heterologous to the fungal host cell (i.e., not native to the fungal host cell). In some additional embodiments, the fungal host cell is engineered to comprise other metabolic pathways that utilize products/intermediates from the pentose phosphate, glycolytic, and/or enthanologenic pathways to produce other desirable products. For example, in some embodiments, the fungal host cell is engineered to comprise a metabolic pathway for the biosynthesis of a fatty alcohol or fatty acid (See e.g., WO 2007/136762, which is incorporated herein by reference). In some embodiments, the fatty alcohol or fatty acid is a C8-C20 fatty acid or fatty alcohol. In some embodiments, the fungal host cell is altered or engineered to overexpress any one or more of the polynucleotides encoding the enzymes in one or more of these metabolic pathways.

In some embodiments, the recombinant fungal host cell of the present invention further comprises genetic modifications in addition to the xylose reductase and/or xylitol dehydrogenase polynucleotide. In some embodiments, in addition to having a xylose reductase and/or xylitol dehydrogenase polynucleotide described herein, the recombinant host cell comprises at least one different recombinant polynucleotide that is capable of conferring a further desired phenotype to the fungal host cell. In some embodiments, the present invention provides a recombinant fungal host cell comprising at least one P. stipitis xylose reductase and/or xylitol dehydrogenase polynucleotide or variant thereof as described herein, and at least one recombinant polynucleotide that encodes a polypeptide which differs from the P. stipitis xylose reductase and/or xylitol dehydrogenase and/or variant(s) thereof, wherein the recombinant polynucleotide imparts a desired phenotype to the fungal host cell. It is contemplated that in some embodiments, the recombinant polynucleotide that is capable of conferring a desired phenotype to the fungal host cell is introduced to the fungal host cell on the same nucleic construct as the xylose reductase and/or xylitol dehydrogenase polynucleotide, or on a separate nucleic acid construct. Nucleic acid constructs of the present invention comprising at least one xylose reductase and xylitol dehydrogenase polynucleotides and at least one further recombinant polynucleotide capable of conferring a desired phenotype to the fungal host cell are also provided by the present invention.

In some embodiments, the recombinant polynucleotide that is capable of conferring a desired phenotype to the fungal host cell is a non-coding polynucleotide (e.g., a regulatory polynucleotide, a coding polynucleotide, or a combination thereof). As described above, exemplary further desired phenotypes include, but are not limited to increased transport of xylose into the host cell, increased xylulose kinase activity, increased flux through the pentose phosphate pathway, decreased sensitivity to catabolite repression, increased tolerance to ethanol, increased tolerance to increased osmolarity, increased tolerance to organic acids, reduced production of by-products, and other like properties related to increasing flux through the pentose phosphate, glycolysis, and/or ethanologenic pathways to produce the desired metabolic product/intermediate at higher levels as compared to the corresponding wild-type host cell. In some embodiments, the desired metabolic product is an alcohol (e.g., ethanol).

In some embodiments, recombinant fungal host cells comprising at least one further polynucleotide capable of conferring a desired phenotype to the fungal host cell comprise at least one polynucleotide encoding a protein known to impact the desired phenotype, wherein the polynucleotide is either native or heterologous to the fungal host cell. In some embodiments, the polynucleotide(s) are operatively linked to its native promoter, while in other embodiments, the polynucleotide is operatively linked to a heterologous promoter (i.e., one not associated with the polynucleotide in the corresponding native gene). In some embodiments, the polynucleotide is overexpressed. In some embodiments, the recombinant fungal host cell comprises multiple copies of the polynucleotide. Suitable polynucleotides include, but are not limited to those that facilitate overexpression of proteins known to have an impact on the desired phenotype. Therefore, in some embodiments, the fungal host cell is altered or engineered to overexpress one or more polynucleotides.

In some embodiments, recombinant polynucleotides that are capable of imparting a desired phenotype to a fungal host cell include, but are not limited to recombinant polynucleotides which encode a xylose or hexose transporter, a xylulose kinase (XKS), an enzyme from the pentose phosphate pathway (See e.g., FIG. 2A), a glycolytic enzyme (i.e., from the metabolic pathway of glycolysis; See e.g., FIG. 2B), and an ethanologenic enzyme (See e.g., FIG. 2C), the regulatory sequences associated with these sequences, and any combination thereof.

Exemplary transporters that find use in the present invention include, but are not limited to GXF1, SUT1 and At6g59250 from Candida intermedia, Pichia supitis, and Arabidopsis thaliana, respectively (See e.g., Runquist et al., 84:37-53 [2010], incorporated herein by reference), HXT4, HXT5, HXT7, GAL2, AGT1, and GXF2, (See e.g., Matsushika et al., Appl. Microbiol. Biotechnol., 84:37-53 [2009]). In some embodiments, overexpression of native S. cerevisiae transporters is desirable, particularly HXT5 and HXT7.

Particularly suitable recombinant polynucleotides include, but are not limited to those that encode: a xylulose kinase (XK); an enzyme from the pentose phosphate pathway (e.g., a ribulose-5-phosphate 3-epimerase (RPE1), a ribose-5-phosphate ketol-isomerase (RKI1), a transketolase (TKL1), a transaldolase (TAL1), etc.); a glycolytic enzyme (e.g., a hexokinase (HXK1/HXK2), a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pyruvate kinase (PVK2), etc.; and an ethanologenic enzyme (e.g., a pyruvate decarboxylase, an alcohol dehydrogenase, etc.).

Exemplary regulatory polynucleotides include promoters, enhancer, terminator, and other regulatory elements that function to improve the expression of polynucleotides in a fungal host cell, particularly, a yeast host cell, as described above.

In some embodiments, recombinant host cells of the present invention comprise one or more native genes deleted from its genome. In some embodiments, the deletion(s) cause removal or diminishment of a biological activity that is otherwise exhibited by the fungal host cell. In some embodiments, the cumulative effect of the deletion(s) also leads to an improvement in a phenotype of the fungal host cell. Any suitable method for deleting gene finds use in the present invention. There are numerous methods well known in the art.

For example, in some embodiments, recombinant host cells of the present invention have certain native genes deleted from the host genome in order to improve the utilization of pentose sugars (e.g., xylose), increase transport of xylose into the host cell, increase xylulose kinase activity, increase flux through the pentose phosphate pathway, decrease sensitivity to catabolite repression, increase tolerance to ethanol/acetate, increase tolerance to increased osmolarity, increase tolerance to organic acids (low pH), reduce production of by-products, and other like properties related to increasing flux through the relevant pathways to produce ethanol and other desired metabolic products at higher levels, where comparison is made with respect to the corresponding host cell without the deletion(s). Genes targeted for deletion include, but are not limited to genes encoding enzymes in the pentose phosphate pathway, a glycolytic enzyme, and/or an ethanologenic enzyme.

In some embodiments, other genes are targeted for deletion, including but not limited to those encoding aldose reductase (GRE3) (See e.g., Matsushika et al., Appl. Microbiol. Biotechnol., 84:37-53 [2009]), sorbitol dehydrogenases (SOR1/SOR2), a glutamate dehydrogenase (GDH1), a 6-phosphogluconate dehydrogenase (GND), a glucose-5-phosphate dehydrogenase (ZWF1), and any enzyme for which its deletion is known in the art to improve the utilization of a pentose sugar, decrease by-product formation, and/or increase the ethanol yield of a fungal host cell. The genes encoding these enzymes in many fungi are known in the art. Those having ordinary skill in the art appreciate that additional genes encoding these enzymes can be readily identified by microarray analysis (See e.g., Sedlak et al., Yeast 21:671-684 [2004]), metabolic flux analysis (See e.g., Sonderegger et al., Appl. Environ. Microbiol., 70(4):2307-2317 [2004]), in silico modeling (See e.g Hjersted et al., Biotechnol. Bioengineer. 97(5):1190-1204 [2007]), chemogenomics (See e.g Teixeira et al., Appl. Environ. Microbiol., 75(18):5761-5772 [2009]), and other well known methods.

In some embodiments, the host cells employed in the practice of the present invention are mutagenized and/or evolved to exhibit further desired phenotypes, for example, further improvement in the utilization of pentose sugars (e.g., xylose, arabinose, etc.), increased transport of xylose into the host cell, increased xylulose kinase activity, increased flux through the pentose phosphate pathway, decreased sensitivity to catabolite repression, increased tolerance to ethanol/acetate, increased tolerance to increased osmolarity, increased tolerance to organic acids (low pH), reduced production of by-products, and other like properties related to increasing flux through the pentose phosphate and glycolysis pathways to produce a desired metabolic product/intermediate at higher levels. In some embodiments, the desired metabolic product is an alcohol (e.g., ethanol). In some embodiments, the host cells are mutagenized and/or evolved using known methods either prior to or after transformation with the xylose reductase and/or xylitol dehydrogenase polynucleotide. These methods include, but are not limited to classical mutagenesis, whole genome shuffling, evolutionary engineering methods, which employ screening and/or selection methods, or any combination of such well known methods.

Classical mutagenesis methods include, but are not limited to treatment of the host cell with a mutagen such as a chemical mutagen or irradiation exposure (e.g., ultraviolet or gamma-irradiation). Whole genome shuffling methods involving, for example, recombination of genomic DNA between native genomic DNA sequences and/or variants thereof, can be facilitated by sexual mating, protoplast fusion methods and other methods well known in the art (See e.g., WO 98/31837 and WO 2000/04190, incorporated herein by reference). These methods are coupled with screening and/or selection methods to identify altered fungal host cells that exhibit the desired phenotype. For example, such methods find use in altering or engineering a fungal host cell to overexpress one or more desired polynucleotides.

Mutagenesis may be performed in accordance with any of the techniques known in the art, including random and site-specific mutagenesis. Directed evolution can be performed with any of the techniques known in the art to screen for improved promoter variants including shuffling. Mutagenesis and directed evolution methods are well known in the art (See e.g., U.S. Pat. Nos. 5,605,793, 5,830,721, 6,132,970, 6,420,175, 6,277,638, 6,365,408, 6,602,986, 7,288,375, 6,287,861, 6,297,053, 6,576,467, 6,444,468, 5,811,238, 6,117,679, 6,165,793, 6,180,406, 6,291,242, 6,995,017, 6,395,547, 6,506,602, 6,519,065, 6,506,603, 6,413,774, 6,573,098, 6,323,030, 6,344,356, 6,372,497, 7,868,138, 5,834,252, 5,928,905, 6,489,146, 6,096,548, 6,387,702, 6,391,552, 6,358,742, 6,482,647, 6,335,160, 6,653,072, 6,355,484, 6,03,344, 6,319,713, 6,613,514, 6,455,253, 6,579,678, 6,586,182, 6,406,855, 6,946,296, 7,534,564, 7,776,598, 5,837,458, 6,391,640, 6,309,883, 7,105,297, 7,795,030, 6,326,204, 6,251,674, 6,716,631, 6,528,311, 6,287,862, 6,335,198, 6,352,859, 6,379,964, 7,148,054, 7,629,170, 7,620,500, 6,365,377, 6,358,740, 6,406,910, 6,413,745, 6,436,675, 6,961,664, 7,430,477, 7,873,499, 7,702,464, 7,783,428, 7,747,391, 7,747,393, 7,751,986, 6,376,246, 6,426,224, 6,423,542, 6,479,652, 6,319,714, 6,521,453, 6,368,861, 7,421,347, 7,058,515, 7,024,312, 7,620,502, 7,853,410, 7,957,912, 7,904,249, and all related US and non-US counterparts; Ling et al., Anal. Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of which are incorporated herein by reference).

Evolutionary engineering can be done by prolonged cultivation and selection of strains under desired conditions through chemostat, turbidostat or batch cultures. Evolutionary engineering methods can be practiced under either aerobic or anaerobic conditions. Selection strategies can be optimized by varying culture conditions, for example, carbon source, nitrogen source, aeration, pH and temperature. Methods for evolutionary engineering are well known in the art (See e.g., Wisselink et al., Appl. Environ. Microbiol., 75(4):907-914 [2009]; Kuyper et al., FEMS Yeast Res., 5:399-409 [2005]; and Sauer, Adv. Biochem. Engineer. Biotechnol., 73:129-169 [2001], all of which are incorporated herein by reference).

Therefore, in some embodiments, the recombinant fungal host cell comprising a xylose reductase and/or xylitol dehydrogenase polynucleotide exhibits an improved phenotype relative to the corresponding fungal host cell without the xylose reductase and/or xylitol dehydrogenase polynucleotide.

In some embodiments, whole genome shuffling and evolutionary engineering methods result in increased copy number of xylose reductase and/or xylitol dehydrogenase and/or xylulokinase from two to fifty copies. It is not intended that the present invention be limited to any particular copy number of xylose reductase and/or xylitol dehydrogenase genes and/or xylulokinase, as any suitable number of copies finds use in the present invention. In some embodiments, the nucleic acid constructs comprise any combination of wild-type and/or variant xylose reductase and/or xylitol dehydrogenase and/or xylulokinase. Furthermore, any suitable method of increasing copies of either xylose reductase and/or xylitol dehydrogenase and/or xylulokinase finds use in the present invention.

In some embodiments, the improved phenotype comprises further improvement in the utilization of pentose sugars (e.g., xylose, arabinose, etc.), increased transport of xylose into the host cell, increased xylulose kinase activity, increased flux through the pentose phosphate pathway, decreased sensitivity to catabolite repression, increased tolerance to ethanol/acetate, increased tolerance to increased osmolarity, increased tolerance to organic acids (low pH), and reduced production of by products, or other properties.

Fermentation

The present invention provides processes for producing fermentation products, wherein the method comprises: (a) providing the recombinant fungal cell of the present invention; (b) providing a fermentation medium comprising xylose; (c) contacting the fermentation medium with the recombinant fungal cell under conditions suitable for generating the fermentation product; and optionally (d) recovering the fermentation product. In some embodiments, the fermentation product is an alcohol (e.g., ethanol, butanol, etc.), a fatty alcohol (e.g., a C8-C20 fatty alcohol), a fatty acid (e.g., a C8-C20 fatty acid), lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propanediol, ethylene, glycerol, and/or a β-lactam (e.g., cephalosporin). However, it is contemplated that other fermentation products will be produced using the methods of the present invention.

In some embodiments, the fermentation medium is feedstock from a cellulosic saccharification process and/or feedstock from a hemicellulose pre-treatment process. Such feedstocks include, but are not limited to carbohydrates (e.g., lignocellulose, xylans, cellulose, starch, etc.), other sugars (e.g., glucose, xylose, arabinose, etc.), and other compositions. Compositions of fermentation media suitable for the growth of yeast and filamentous fungi are well known in the art and there are various reference texts that provide recipes for these media.

Fermentation conditions suitable for generating desired fermentation products are well known in the art and any suitable method finds use in the present invention. In some embodiments, the fermentation process is carried out under aerobic or microaerobic (i.e., where the concentration of oxygen is less than that in air), or anaerobic conditions. In some embodiments, fermentation is conducted under anaerobic conditions (i.e., no detectable oxygen), or less than about 5, about 2.5, or about 1 mmol/L/h oxygen. In the absence of oxygen, the NADH produced in glycolysis cannot be oxidized by oxidative phosphorylation. Under anaerobic conditions, pyruvate or a derivative thereof may be utilized by the host cell as an electron and hydrogen acceptor in order to generated NAD+. In some embodiments of the present invention, when the fermentation process is carried out under anaerobic conditions, pyruvate may be reduced to a fermentation product such as ethanol, butanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propanediol, ethylene, glycerol, and/or β-lactam (e.g., a cephalosporin).

The fermentation process is typically run at a temperature that is optimal for the recombinant fungal cell. For example, in some embodiments, the fermentation process is performed at a temperature in the range of from about 25° C. to about 42° C. Typically the process is carried out a temperature that is less than about 38° C., less than about 35° C., less than about 33° C., or less than about 38° C., but at least about 20° C., 22° C., or 25° C.

In some embodiments, recombinant host cells of the present invention are grown under batch or continuous fermentation conditions. Classical batch fermentation is a closed system, wherein the composition of the medium is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. A variation of the batch system is a fed-batch fermentation, which also finds use in the present invention. In this variation, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and/or where it is desirable to have limited amounts of substrate in the medium. Batch and fed-batch fermentations are common and well known in the art. Continuous fermentation is an open system where a defined fermentation generally maintains the culture at a constant high density where cells are primarily in log phase growth. Continuous fermentation systems strive to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes, as well as techniques for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

The foregoing and other aspects of the invention may be better understood in connection with the following non-limiting examples.

EXPERIMENTAL

The present invention is described in further detail in the following Examples, which are not in any way intended to limit the scope of the invention as claimed.

In the experimental disclosure below, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and μg (micrograms); L and l (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); ° C. (degrees Centigrade); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); HPLC (high pressure liquid chromatography); MES (2-N-morpholino ethanesulfonic acid); FIOPC (fold improvements over positive control); YPD (10 g/L yeast extract, 20 g/L peptone, and 20 g/L dextrose); SOE-PCR (splicing by overlapping extension PCR); ARS (ARS Culture Collection or NRRL Culture Collection, Peoria, Ill.); Axygen (Axygen, Inc., Union City, Calif.); Lallemand (Lallemand Ethanol Technology, Milwaukee, Wis.); Dual Biosystems (Dual Biosystems AG, Schlieven, Switzerland); Megazyme (Megazyme International Ireland, Ltd., Wicklow, Ireland); Dasgip (Dasgip Biotools, LLC, Shrewsbury, Mass.); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, Mich.); PCRdiagnostics (PCRdiagnostics, by E coli SRO, Slovak Republic); Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); and Bio-Rad (Bio-Rad Laboratories, Hercules, Calif.).

The following sequences find use in the present invention. SEQ ID NO:2 provides the polypeptide sequence of P. stipitis xylose reductase (“XR.1”), while SEQ ID NO:1 provides the native polynucleotide sequence, SEQ ID NO:3 provides one codon optimized DNA sequence (“XR.2”), and SEQ ID NO:4 provides an alternative codon optimized DNA sequence (“XR.3”). SEQ ID NO:6 provides the polypeptide sequence of P. stipitis xylitol dehydrogenase (“XD.1”), while SEQ ID NO:5 provides the native polynucleotide sequence, SEQ ID NO:7 provides one codon optimized DNA sequence (“XD.2”), and SEQ ID NO:8 provides an alternative codon optimized DNA sequence (“XD.3”). SEQ ID NO:10 provides the polypeptide sequence of S. cerevisiae xylulokinase (“XK.1”), SEQ ID NO:9 provides the native polynucleotide sequence, SEQ ID NO:11 provides one codon optimized DNA sequence (“XK.2”), and SEQ ID NO:12 provides an alternative codon optimized DNA sequence (“XK.3”).

(SEQ ID NO: 2) MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQIYRAIKTGYRLFDGAEDYA NEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSD LQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETW KALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQ PRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIA AKHGKSPAQVLLRWSSQRGIAIIPKSNTVPRLLENKDVNSFDLDEQDFA DIAKLDINLRFNDPWDWDKIPIFV (SEQ ID NO: 1) ATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTT TCGGCTGTTGGAAAGTCGACGTCGACACCTGTTCTGAACAGATCTACCG TGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGCC AACGAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTA TCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTA CCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGAC TTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCT TCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGG TAAGGGTGACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGG AAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGATCTATCGGTG TTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTAC CATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAA CCAAGATTGATCGAATTCGCTCAATCCCGTGGTATTGCTGTCACCGCTT ACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGC TTTGAACACTTCTCCATTGTTCGAGAACGAAACTATCAAGGCTATCGCT GCTAAGCACGGTAAGTCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCC AAAGAGGCATTGCCATCATTCCAAAGTCCAACACTGTCCCAAGATTGTT GGAAAACAAGGACGTCAACAGCTTCGACTTGGACGAACAAGATTTCGCT GACATTGCCAAGTTGGACATCAACTTGAGATTCAACGACCCATGGGACT GGGACAAGATTCCTATCTTCGTCTAATAA (SEQ ID NO: 3) ATGCCCTCCATAAAGCTAAACTCTGGTTATGATATGCCTGCCGTTGGAT TCGGTTGTTGGAAAGTAGATGTTGATACCTGCTCAGAACAGATTTATAG GGCTATTAAAACAGGTTACAGGTTGTTCGACGGTGCCGAGGACTACGCG AATGAGAAGTTAGTTGGAGCTGGTGTTAAGAAGGCCATCGACGAGGGCA TTGTTAAGAGGGAGGACCTGTTCTTAACGTCTAAACTGTGGAATAACTA TCACCACCCAGACAATGTTGAAAAAGCCCTGAATAGAACTTTGAGTGAT TTGCAGGTGGATTACGTTGACTTGTTTCTAATCCATTTCCCCGTTACTT TCAAGTTCGTCCCTTTAGAGGAGAAGTATCCACCAGGTTTCTATTGTGG TAAGGGCGACAACTTCGACTATGAAGATGTTCCTATTCTGGAGACCTGG AAGGCTCTGGAGAAACTGGTCAAGGCAGGCAAAATAAGGTCCATAGGCG TCTCTAACTTCCCAGGAGCACTACTGCTTGACTTGTTGAGGGGAGCTAC CATCAAACCTTCAGTCTTGCAAGTTGAACACCACCCCTATTTGCAACAA CCCAGGCTGATAGAGTTTGCTCAATCCAGAGGTATAGCTGTTACTGCAT ACTCTTCCTTTGGACCTCAATCCTTCGTCGAACTTAACCAAGGTAGAGC GTTGAATACTTCCCCCTTGTTCGAGAATGAAACTATCAAGGCTATTGCG GCTAAGCACGGTAAGTCACCAGCTCAAGTGTTACTTAGATGGTCTTCCC AAAGGGGTATCGCAATCATTCCAAAGTCTAACACTGTTCCCAGATTGTT GGAGAATAAAGATGTTAATTCTTTCGATTTGGACGAACAAGACTTTGCA GACATAGCCAAACTAGATATTAATCTGAGATTCAATGATCCCTGGGATT GGGACAAGATACCAATCTTCGTTTAATAA (SEQ ID NO: 4) ATGCCATCTATTAAGTTGAACTCTGGTTACGACATGCCAGCTGTCGGTT TCGGTTGTAAGGTTGACGTTGACACCTGTTCTGAACAAATTTACAGAGC TATTAAGACCGGTTACAGATTGTTCGACGGTGCTGAAGACTACGCTAAC GAAAAGTTGGTTGGTGCTGGTGTCAAGAAGGCTATTGACGAAGGTATTG TCAAGAGAGAAGACTTGTTCTTGACCTCTAAGTTGAACAACTACCACCA CCCAGACAACGTCGAAAAGGCTTTGAACAGAACCTTGTCTGACTTGCAA GTTGACTACGTTGACTTGTTCTTGATTCACTTCCCAGTCACCTTCAAGT TCGTCCCATTGGAAGAAAAGTACCCACCAGGTTTCTACTGTGGTAAGGG TGACAACTTCGACTACGAAGACGTTCCAATTTTGGAAACCAAGGCTTTG GAAAAGTTGGTTAAGGCTGGTAAGATTAGGTCTATTGGTGTTTCTAACT TCCCAGGTGCTTTGTTGTTGGACTTGTTGAGAGGTGCTACCATTAAGCC ATCTGTTTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTG ATTGAGTTCGCTCAATCTAGGGGTATTGCTGTCACCGCTTACTCTTCTT TCGGTCCACAATCTTTCGTCGAATTGAACCAAGGTAGAGCTTTGAACAC CTCTCCATTGTTCGAAAACGAAACCATTAAGGCTATTGCTGCTAAGCAC GGTAAGTCTCCAGCTCAAGTCTTGTTGAGGTCTTCTCAAAGAGGTATTG CTATTATTCCAAAGTCTAACACCGTCCCAAGATTGTTGGAAAACAAGGA CGTTAACTCTTTCGACTTGGACGAACAAGACTTCGCTGACATTGCTAAG TTGGACATTAACTTGAGATTCAACGACCCAGACGACAAGATTCCAATTT TCGTTTAATAA (SEQ ID NO: 6) MTANPSLVLNKIDDISFETYDAPEISEPTDVLVQVKKTGICGSDIHFYA HGRIGNFVLTKPMVLGHESAGTVVQVGKGVTSLKVGDNVAIEPGIPSRF SDEYKSGHYNLCPHMAFAATPNSKEGEPNPPGTLCKYFKSPEDFLVKLP DHVSLELGALVEPLSVGVHASKLGSVAFGDYVAVFGAGPVGLLAAAVAK TFGAKGVIVVDIFDNKLKMAKDIGAATHTFNSKTGGSEELIKAFGGNVP NVVLECTGAEPCIKLGVDAIAPGGRFVQVGNAAGPVSFPITVFAMKELT LFGSFRYGFNDYKTAVGIFDTNYQNGRENAPIDFEQLITHRYKFKDAIE AYDLVRAGKGAVKCLIDGPE (SEQ ID NO: 5) ATGACTGCTAACCCTTCCTTGGTGTTGAACAAGATCGACGACATTTCGT TCGAAACTTACGATGCCCCAGAAATCTCTGAACCTACCGATGTCCTCGT CCAGGTCAAGAAAACCGGTATCTGTGGTTCCGACATCCACTTCTACGCC CATGGTAGAATCGGTAACTTCGTTTTGACCAAGCCAATGGTCTTGGGTC ACGAATCCGCCGGTACTGTTGTCCAGGTTGGTAAGGGTGTCACCTCTCT TAAGGTTGGTGACAACGTCGCTATCGAACCAGGTATTCCATCCAGATTC TCCGACGAATACAAGAGCGGTCACTACAACTTGTGTCCTCACATGGCCT TCGCCGCTACTCCTAACTCCAAGGAAGGCGAACCAAACCCACCAGGTAC CTTATGTAAGTACTTCAAGTCGCCAGAAGACTTCTTGGTCAAGTTGCCA GACCACGTCAGCTTGGAACTCGGTGCTCTTGTTGAGCCATTGTCTGTTG GTGTCCACGCCTCTAAGTTGGGTTCCGTTGCTTTCGGCGACTACGTTGC CGTCTTTGGTGCTGGTCCTGTTGGTCTTTTGGCTGCTGCTGTCGCCAAG ACCTTCGGTGCTAAGGGTGTCATCGTCGTTGACATTTTCGACAACAAGT TGAAGATGGCCAAGGACATTGGTGCTGCTACTCACACCTTCAACTCCAA GACCGGTGGTTCTGAAGAATTGATCAAGGCTTTCGGTGGTAACGTGCCA AACGTCGTTTTGGAATGTACTGGTGCTGAACCTTGTATCAAGTTGGGTG TTGACGCCATTGCCCCAGGTGGTCGTTTCGTTCAAGTCGGTAACGCTGC TGGTCCAGTCAGCTTCCCAATCACCGTTTTCGCCATGAAGGAATTGACT TTGTTCGGTTCTTTCAGATACGGATTCAACGACTACAAGACTGCTGTTG GAATCTTTGACACTAACTACCAAAACGGTAGAGAAAATGCTCCAATTGA CTTTGAACAATTGATCACCCACAGATACAAGTTCAAGGACGCTATTGAA GCCTACGACTTGGTCAGAGCCGGTAAGGGTGCTGTCAAGTGTCTCATTG ACGGCCCTGAGTAATAA (SEQ ID NO: 7) ATGACCGCTAATCCCTCTCTTGTTTTGAATAAGATTGACGACATTTCTT TTGAAACTTACGATGCTCCCGAAATTAGCGAACCCACAGACGTTTTAGT TCAAGTTAAAAAAACTGGTATCTGCGGTTCTGACATCCACTTCTACGCT CATGGAAGGATCGGCAACTTCGTCTTAACAAAGCCAATGGTTCTGGGTC ATGAAAGCGCGGGTACTGTTGTTCAAGTCGGTAAAGGTGTTACTTCACT GAAGGTTGGTGATAACGTCGCAATCGAGCCCGGTATTCCATCTAGGTTC AGTGATGAGTACAAATCTGGTCACTACAACCTGTGTCCACACATGGCAT TTGCTGCTACTCCCAATTCTAAAGAGGGTGAACCAAACCCACCAGGAAC TCTATGTAAGTACTTCAAATCTCCAGAAGACTTCCTGGTTAAGTTACCC GATCATGTTTCTTTGGAGTTGGGTGCTTTGGTCGAGCCACTATCTGTTG GGGTCCATGCTAGTAAATTAGGCTCCGTTGCATTTGGCGATTACGTTGC TGTTTTTGGTGCTGGTCCAGTAGGATTACTGGCTGCCGCTGTCGCTAAG ACATTTGGTGCCAAGGGTGTGATTGTCGTTGATATATTTGACAACAAGC TGAAGATGGCCAAAGACATAGGTGCCGCTACACATACCTTCAACTCCAA GACGGGAGGTAGTGAAGAATTGATCAAAGCCTTCGGTGGTAATGTACCA AATGTTGTCTTGGAATGTACTGGGGCTGAACCATGTATTAAGCTAGGTG TTGATGCCATCGCACCAGGTGGTAGATTCGTGCAAGTTGGTAATGCTGC TGGTCCCGTGTCCTTTCCCATAACAGTGTTCGCTATGAAAGAACTTACT TTGTTTGGTTCATTTCGTTATGGTTTCAACGACTATAAGACAGCCGTGG GTATCTTTGATACTAACTACCAGAACGGTAGAGAGAATGCTCCCATTGA CTTTGAACAGCTTATCACGCACAGATACAAATTCAAAGACGCCATTGAA GCCTACGACCTAGTAAGAGCAGGTAAAGGGGCTGTCAAGTGTTTGATTG ATGGTCCAGAATAATAA (SEQ ID NO: 8) ATGACCGCTAACCCATCTTTGGTCTTGAACAAGATTGACGACATTTCTT TCGAAACCTACGACGCTCCAGAAATTTCTGAACCAACCGACGTTTTGGT TCAAGTCAAGAAGACCGGTATTTGTGGTTCTGACATTCACTTCTACGCT CACGGTAGAATTGGTAACTTCGTCTTGACCAAGCCAATGGTCTTGGGTC ACGAATCTGCTGGTACCGTTGTTCAAGTCGGTAAGGGTGTCACCTCTTT GAAGGTCGGTGACAACGTCGCTATTGAACCAGGTATTCCAAGTAGATTC TCTGACGAATACAAGTCTGGTCACTACAACTTGTGTCCACACATGGCTT TCGCTGCTACCCCAAACTCTAAGGAAGGTGAACCAAACCCACCAGGTAC CTTGTGTAAGTACTTCAAGTCTCCAGAAGACTTCTTGGTTAAGTTGCCA GACCACGTTTCTTTGGAATTGGGTGCTTTGGTTGAACCATTGTCTGTTG GTGTTCACGCTTCTAAGTTGGGTTCTGTTGCTTTCGGTGACTACGTTGC TGTTTTCGGTGCTGGTCCAGTTGGTTTGTTGGCTGCTGCTGTTGCTAAG ACCTTCGGTGCTAAGGGTGTCATTGTCGTTGACATTTTCGACAACAAGT TGAAGATGGCTAAGGACATTGGTGCTGCTACCCACACCTTCAACTCTAA GACCGGTGGTTCTGAAGAATTGATTAAGGCTTTCGGTGGTAACGTCCCA AACGTCGTCTTGGAATGTACCGGTGCTGAACCATGTATTAAGTTGGGTG TTGACGCTATTGCTCCAGGTGGTAGATTCGTCCAAGTTGGTAACGCTGC TGGTCCAGTTTCTTTCCCAATTACCGTTTTCGCTATGAAGGAATTGACC TTGTTCGGTTCTTTCAGATACGGTTTCAACGACTACAAGACCGCTGTTG GTATTTTCGACACCAACTACCAAAACGGTAGAGAAAACGCTCCAATTGA CTTCGAACAATTGATTACCCACAGATACAAGTTCAAGGACGCTATTGAA GCTTACGACTTGGTTAGAGCTGGTAAGGGTGCTGTTAAGTGTTTGATTG ACGGTCCAGAATAATAA (SEQ ID NO: 9) ATGTTGTGTTCAGTAATTCAGAGACAGACAAGAGAGGTTTCCAACACAA TGTCTTTAGACTCATACTATCTTGGGTTTGATCTTTCGACCCAACAACT GAAATGTCTCGCCATTAACCAGGACCTAAAAATTGTCCATTCAGAAACA GTGGAATTTGAAAAGGATCTTCCGCATTATCACACAAAGAAGGGTGTCT ATATACACGGCGACACTATCGAATGTCCCGTAGCCATGTGGTTAGAGGC TCTAGATCTGGTTCTCTCGAAATATCGCGAGGCTAAATTTCCATTGAAC AAAGTTATGGCCGTCTCAGGGTCCTGCCAGCAGCACGGGTCTGTCTACT GGTCCTCCCAAGCCGAATCTCTGTTAGAGCAATTGAATAAGAAACCGGA AAAAGATTTATTGCACTACGTGAGCTCTGTAGCATTTGCAAGGCAAACC GCCCCCAATTGGCAAGACCACAGTACTGCAAAGCAATGTCAAGAGTTTG AAGAGTGCATAGGTGGGCCTGAAAAAATGGCTCAATTAACAGGGTCCAG AGCCCATTTTAGATTTACTGGTCCTCAAATTCTGAAAATTGCACAATTA GAACCAGAAGCTTACGAAAAAACAAAGACCATTTCTTTAGTGTCTAATT TTTTGACTTCTATCTTAGTGGGCCATCTTGTTGAATTAGAGGAGGCAGA TGCCTGTGGTATGAACCTTTATGATATACGTGAAAGAAAATTCAGTGAT GAGCTACTACATCTAATTGATAGTTCTTCTAAGGATAAAACTATCAGAC AAAAATTAATGAGAGCACCCATGAAAAATTTGATAGCGGGTACCATCTG TAAATATTTTATTGAGAAGTACGGTTTCAATACAAACTGCAAGGTCTCT CCCATGACTGGGGATAATTTAGCCACTATATGTTCTTTACCCCTGCGGA AGAATGACGTTCTCGTTTCCCTAGGAACAAGTACTACAGTTCTTCTGGT CACCGATAAGTATCACCCCTCTCCGAACTATCATCTTTTCATTCATCCA ACTCTGCCAAACCATTATATGGGTATGATTTGTTATTGTAATGGTTCTT TGGCAAGGGAGAGGATAAGAGACGAGTTAAACAAAGAACGGGAAAATAA TTATGAGAAGACTAACGATTGGACTCTTTTTAATCAAGCTGTGCTAGAT GACTCAGAAAGTAGTGAAAATGAATTAGGTGTATATTTTCCTCTGGGGG AGATCGTTCCTAGCGTAAAAGCCATAAACAAAAGGGTTATCTTCAATCC AAAAACGGGTATGATTGAAAGAGAGGTGGCCAAGTTCAAAGACAAGAGG CACGATGCCAAAAATATTGTAGAATCACAGGCTTTAAGTTGCAGGGTAA GAATATCTCCCCTGCTTTCGGATTCAAACGCAAGCTCACAACAGAGACT GAACGAAGATACAATCGTGAAGTTTGATTACGATGAATCTCCGCTGCGG GACTACCTAAATAAAAGGCCAGAAAGGACTTTTTTTGTAGGTGGGGCTT CTAAAAACGATGCTATTGTGAAGAAGTTTGCTCAAGTCATTGGTGCTAC AAAGGGTAATTTTAGGCTAGAAACACCAAACTCATGTGCCCTTGGTGGT TGTTATAAGGCCATGTGGTCATTGTTATATGACTCTAATAAAATTGCAG TTCCTTTTGATAAATTTCTGAATGACAATTTTCCATGGCATGTAATGGA AAGCATATCCGATGTGGATAATGAAAATTGGGATCGCTATAATTCCAAG ATTGTCCCCTTAAGCGAACTGGAAAAGACTCTCATCTAA (SEQ ID NO: 10) MLCSVIQRQTREVSNTMSLDSYYLGFDLSTQQLKCLAINQDLKIVHSET VEFEKDLPHYHTKKGVYIHGDTIECPVAMWLEALDLVLSKYREAKFPLN KVMAVSGSCQQHGSVYWSSQAESLLEQLNKKPEKDLLHYVSSVAFARQT APNWQDHSTAKQCQEFEECIGGPEKMAQLTGSRAHFRFTGPQILKIAQL EPEAYEKTKTISLVSNFLTSILVGHLVELEEADACGMNLYDIRERKFSD ELLHLIDSSSKDKTIRQKLMRAPMKNLIAGTICKYFIEKYGFNTNCKVS PMTGDNLATICSLPLRKNDVLVSLGTSTTVLLVTDKYHPSPNYHLFIHP TLPNHYMGMICYCNGSLARERIRDELNKERENNYEKTNDWTLFNQAVLD DSESSENELGVYFPLGEIVPSVKAINKRVIFNPKTGMIEREVAKFKDKR HDAKNIVESQALSCRVRISPLLSDSNASSQQRLNEDTIVKFDYDESPLR DYLNKRPERTFFVGGASKNDAIVKKFAQVIGATKGNFRLETPNSCALGG CYKAMWSLLYDSNKIAVPFDKFLNDNFPWHVMESISDVDNENWDRYNSK IVPLSELEKTLI (SEQ ID NO: 11) ATGCTGTGCTCCGTTATACAAAGGCAAACAAGAGAAGTATCCAACACTA TGTCTTTAGATAGTTATTATCTAGGATTCGATTTAAGTACACAACAATT GAAATGTCTTGCTATAAACCAGGATCTAAAGATCGTCCATTCCGAAACT GTCGAGTTCGAGAAGGACTTACCACATTATCACACCAAGAAAGGCGTCT ACATTCATGGTGACACCATCGAATGCCCAGTTGCTATGTGGTTAGAAGC CCTGGATCTTGTCCTGTCCAAATATAGGGAGGCAAAGTTCCCACTGAAC AAGGTCATGGCTGTTTCCGGTTCTTGTCAGCAGCATGGCTCCGTCTACT GGTCATCACAGGCTGAATCTCTGTTAGAACAACTGAACAAGAAGCCAGA GAAGGACCTGTTACACTACGTCTCCTCTGTTGCATTTGCCAGACAAACT GCTCCTAATTGGCAAGACCATTCCACTGCTAAACAATGTCAGGAGTTCG AAGAGTGTATTGGTGGACCAGAGAAAATGGCCCAGTTAACTGGTTCCCG TGCTCATTTCAGGTTCACAGGCCCACAAATCCTGAAGATTGCTCAGTTA GAACCAGAGGCTTATGAAAAGACTAAGACCATCTCTTTGGTCTCTAATT TCTTAACTTCCATTCTGGTTGGTCACTTGGTCGAACTGGAAGAAGCTGA TGCGTGTGGTATGAACCTGTACGACATCCGTGAGAGGAAGTTCTCTGAC GAACTGCTGCATCTTATCGACTCCTCCTCTAAGGACAAGACCATCAGGC AGAAACTGATGAGGGCACCAATGAAGAACCTGATTGCCGGTACTATTTG CAAGTACTTCATCGAAAAGTATGGCTTCAACACCAACTGCAAAGTCTCC CCTATGACTGGCGATAACCTAGCCACCATTTGTAGCTTGCCCTTAAGAA AAAACGATGTTCTTGTGTCTTTGGGTACTTCCACAACCGTCTTGTTGGT TACCGACAAATATCACCCTTCACCAAACTACCACCTGTTCATCCACCCG ACGTTGCCTAACCACTACATGGGCATGATCTGCTACTGCAATGGCAGTT TAGCAAGGGAAAGGATAAGGGACGAGTTGAACAAGGAGAGGGAGAACAA CTACGAGAAGACCAACGATTGGACCCTGTTCAACCAAGCTGTCCTGGAT GATAGCGAATCCTCCGAGAATGAACTGGGCGTTTACTTTCCACTAGGCG AGATCGTTCCATCTGTCAAGGCCATCAACAAGAGAGTAATCTTCAACCC CAAGACTGGCATGATCGAAAGGGAAGTCGCCAAGTTCAAGGACAAGAGA CATGACGCCAAGAACATCGTTGAATCTCAAGCCTTATCTTGCCGTGTTA GGATTTCTCCCCTACTAAGCGACTCCAATGCTTCTTCCCAGCAACGTTT GAACGAGGATACGATTGTTAAATTCGACTACGACGAGAGTCCATTGAGA GACTACTTGAACAAACGTCCTGAGAGGACATTCTTTGTTGGTGGCGCAT CCAAGAACGATGCTATTGTTAAGAAGTTTGCTCAGGTCATAGGAGCAAC CAAAGGTAACTTTCGTTTAGAAACTCCAAACTCATGCGCTTTAGGTGGT TGCTACAAGGCTATGTGGTCTTTGTTGTATGATAGCAATAAAATCGCTG TTCCTTTCGACAAGTTCCTAAACGATAACTTCCCTTGGCACGTCATGGA ATCCATCAGCGATGTAGACAACGAGAATTGGGATAGATACAATTCTAAA ATAGTTCCCTTGTCTGAGTTAGAGAAGACCTTGATTTAATAA (SEQ ID NO: 12) ATGTTGTGTTCTGTCATTCAAAGACAAACCAGAGAAGTTTCTAACACCA TGTCTTTGGACTCTTACTACTTGGGTTTCGACTTGTCTACCCAACAATT GAAGTGTTTGGCTATTAACCAAGACTTGAAGATTGTCCACTCTGAAACC GTTGAGTTCGAAAAGGACTTGCCACACTACCACACCAAGAAGGGTGTCT ACATTCACGGTGACACCATTGAATGTCCAGTCGCTATGTTGGAAGCTTT GGACTTGGTTTTGTCTAAGTACAGAGAAGCTAAGTTCCCATTGAACAAG GTCATGGCTGTCTCTGGTTCTTGTCAACAACACGGTTCTGTCTACTCTT CTCAAGCTGAATCTTTGTTGGAACAATTGAACAAGAAGCCAGAAAAGGA CTTGTTGCACTACGTCTCTTCTGTCGCTTTCGCTAGACAAACCGCTCCA AACCAAGACCACTCTACCGCTAAGCAATGTCAAGAGTTCGAAGAATGTA TTGGTGGTCCAGAAAAGATGGCTCAATTGACCGGTAGTAGAGCACACTT CAGATTCACCGGTCCACAAATTTTGAAGATTGCTCAATTGGAACCAGAA GCTTACGAAAAGACCAAGACCATTTCTTTGGTCTCTAACTTCTTGACCT CTATTTTGGTCGGTCACTTGGTCGAATTGGAAGAAGCTGACGCTTGTGG TATGAACTTGTACGACATTAGAGAAAGAAAGTTCTCTGACGAATTGTTG CACTTGATTGACTCTTCTTCTAAGGACAAGACCATTAGACAAAAGTTGA TGAGGGCTCCAATGAAGAACTTGATTGCTGGTACCATTTGTAAGTACTT CATTGAAAAGTACGGTTTCAACACCAACTGTAAGGTCTCTCCAATGACC GGTGACAACTTGGCTACCATTTGTTCTTTGCCATTGAGAAAGAACGACG TTTTGGTTTCTTTGGGTACCTCTACCACCGTCTTGTTGGTTACCGACAA GTACCACCCATCTCCAAACTACCACTTGTTCATTCACCCAACCTTGCCA AACCACTACATGGGTATGATTTGTTACTGTAACGGTTCTTTGGCTAGAG AAAGAATTAGAGACGAATTGAACAAGGAAAGAGAAAACAACTACGAAAA GACCAACGACACCTTGTTCAACCAAGCTGTTTTGGACGACTCTGAATCT TCTGAAAACGAATTGGGTGTCTACTTCCCATTGGGTGAAATTGTTCCAT CTGTCAAGGCTATTAACAAGAGAGTCATTTTCAACCCAAAGACCGGTAT GATTGAAAGAGAAGTCGCTAAGTTCAAGGACAAGAGACACGACGCTAAG AACATTGTCGAATCTCAAGCTTTGTCTTGTAGAGTTAGAATTTCTCCAT TGTTGTCTGACTCTAACGCTTCTTCTCAACAAAGATTGAACGAAGACAC CATTGTCAAGTTCGACTACGACGAATCTCCATTGAGAGACTACTTGAAC AAGAGACCAGAAAGAACCTTCTTCGTTGGTGGTGCTTCTAAGAACGACG CTATTGTTAAGAAGTTCGCTCAAGTCATTGGTGCTACCAAGGGTAACTT CAGATTGGAAACCCCAAACTCTTGTGCTTTGGGTGGTTGTTACAAGGCT ATGTCTTTGTTGTACGACTCTAACAAGATTGCTGTTCCATTCGACAAGT TCTTGAACGACAACTTCCCACACGTCATGGAATCTATTTCTGACGTTGA CAACGAAAACGACAGATACAACTCTAAGATTGTTCCATTGTCTGAATTG GAAAAGACCTTGATTTAATAA

Example 1 Construction of Plasmid PLS2802 with XR, XD and XK Genes

Xylose reductase from Pichis stipitis (XR.2; SEQ ID NO:3), xylitol dehydrogenase from Pichia stipitis (XD.2; SEQ ID NO:7), and xylulose kinase (XK.2; SEQ ID NO:11) from Saccharomyces cerevisiae were synthesized with codon optimization for expression in yeast. The genes were synthesized with the following 5′ and 3′ flanks to introduce specific restriction enzyme sites:

(SEQ ID NO: 13) XR 5′ GGATCCCAAACAAA; and (SEQ ID NO: 14) 3′ CATATG to introduce 5′-BamHI and 3′-NdeI; (SEQ ID NO: 15) XD 5′ ACTAGTCAAACAAA; and (SEQ ID NO: 16) 3′-GACGTC to introduce 5′SpeI and 3′ AatII; and (SEQ ID NO: 17) XK 5′ GCGGCCGCCAAACAAA; and (SEQ ID NO: 18) 3′ CTCGAG to introduce 5′ NotI and 3′ XhoI

The yeast vector p427TEF (Dualsystems Biotech AG) was used for gene expression. The vector contains a kanamycin resistance gene that allows for selection in yeast, an ampicillin resistance gene that allows for selection in E. coli, and a 2 micron origin of replication that allows for propagation of plasmids in high copy numbers in yeast. For cloning the pathway genes, p427TEF was digested with SacI and XhoI restriction enzymes. The larger fragment (6235 bp) was ligated with an oligomer of the following sequence, 5′GAGCTCACGGATCCGTCATATGCTAGATCTCTGAATTCTTACTAGTTCGACGTCTACCTAG GCAGTCGACACGCGGCCGCTTCTCGAG 3′ (SEQ ID NO:19) to introduce a new multiple cloning site (MCS) with desired restriction sites. Using the new MCS, the TEF1 promoter of S. cerevisiae was re-introduced in the vector using SacI/BamHI restriction sites to create PLS1567. To clone all 3 genes into one vector, additional promoters (Adh1p, GPDp) and terminators (Adh2t, Adh1t) were cloned into PLS1567 using yeast recombinational cloning using methods commonly used in the art. The promoters and terminators were each amplified using the primer sets shown in Table 1-1.

TABLE 1-1 Primers Used to Amplify Yeast Promoters and Terminators for PLS1448 Construction Cassette Primer 1 Primer 2 ADH2 GCA TAG CAA TCT AAT CTA AGT TTG TAT GTA CCT GTC TGA ATT terminator TTT GGA TCC GTC ATA TGG CGG CAG AGA TCT AGC CCT GAG AAA ATC TCT TAT GTC TTT ACG CTA TAT GAG GGT (SEQ ID NO: 20) (SEQ ID NO: 21) ADH1 ACC CTC ATA TAG TTT CTC AGG TCA TAA ATC ATA AGA AAT TCG promoter GCT AGA TCT CTG AAT TCA GAC CGA CGTCGA ACT AGT TGT ATA AGG TAC ATA CAA TGA GAT AGT TGA TTG TAT GC (SEQ ID NO: 22) (SEQ ID NO: 23) ADH1 GCA TAC AAT CAA CTA TCT CAT TGA TAA ACT CGA AGT CGA CTG terminator ATA CAA CTA GTT CGA CGT CGC CCT AGG CAT GCC GGT AGA GGT GAA TTT CTT ATG ATT TAT GA GTG GT (SEQ ID NO: 24) (SEQ ID NO: 25) GPD ACC ACA CCT CTA CCG GCA TGC GCG TGA CAT AAC TAA TTA CAT promoter CTA GGC AGT CGA CTT CGA GTT GAC TCG AGA AGC GGC CGC TTT TAT CA GTT TGT TTA TGT GTGT (SEQ ID NO: 26) (SEQ ID NO: 27)

SOE-PCR was used to combine the ADH2 terminator with the ADH1 promoter, and the ADH1 terminator with the GPD1 promoter. An internal AatII site in the ADH2 terminator was removed by changing its sequence from GACGTC (SEQ ID NO:28) to GATGTC (SEQ ID NO:29). For yeast recombinational cloning, PLS1567 was digested with BamHI/XhoI, and a 3:1 mass ratio of each insert to digested PLS1567 was used. The transformants were selected on culture plates containing G418. The resulting plasmid (PLS1448) with the 3 promoters and terminators was confirmed by sequencing. The 2 micron origin in PLS1448 was replaced with the CEN6/ARS4 origin of replication to yield PLS1565 with lower copy number and greater stability.

XR.2, XD.2 and XK.2 genes were then cloned into PLS1565 using yeast recombinational cloning. To amplify all the pieces, the primer sets provided in Table 1-2 were used.

TABLE 1-2 Primers Used to Amplify Cassettes for PLS2802 Construction Cassette Primer 1 Primer 2 PS.XR.2 CTT GCT CAT TAG AAA GAA AGC CTA TAA ATC GTA AAG ACA TAA ATA GCA ATC TAA TCT AAG TTT GAG ATC CGC CAT ATG TTA TTA TGG ATC CCA AAC AAA ATG CCC AAC GAA GAT TGG TAT CTT G TCC A (SEQ ID NO: 31) (SEQ ID NO: 30) ADH2t- CAA GAT ACC AAT CTT CGT TTA GAG GGA TTA GCG GTC ATT TTG ADH 1p ATA ACA TAT GGC GGA TCT CTT TTT GAC TAG TTG TAT ATG AGA ATG TCT TTA CGA TTT ATA G TAG TTG ATT GTA TGC TTG GTA (SEQ ID NO: 32) (SEQ ID NO: 33) PS.XD.2 TAC CAA GCA TAC AAT CAA CTA TAA TAA AAA TCA TAA ATC ATA TCT CAT ATA CAA CTA GTC AAA AGA AAT TCG CGA CGT CTT ATT CAA AAT GAC CGC TAA TCC CTC ATT CTG GAC CAT CAA TCA (SEQ ID NO: 34) (SEQ ID NO: 35) ADH1t- TGA TTG ATG GTC CAG AAT AAT TTT GTA TAA CGG AGC ACA GCA GPDp AAG ACG TCG CGA ATT TCT TAT TTT TGT TTG GCG GCC GCT TTG GAT TTA TGA TTT TTA TTA TTT GTT TAT GTG TGT TTA T (SEQ ID NO: 36) (SEQ ID NO: 37) SC.XK.2 ATA AAC ACA CAT AAA CAA ACA GGG GAG GGC GTG AAT GTA AGC AAG CGG CCG CCA AAC AAA ATG GTG ACA TAA CTA ATT ACA TGA CTG TGC TCC GTT ATA CAA A CTC GAG TTA TTA AAT CAA GGT (SEQ ID NO: 38) CTT CTC T (SEQ ID NO: 39)

For yeast recombinational cloning, PLS1565 was digested with BamHI/XhoI and gel purified. Then, a 3:1 mass ratio of each insert:digested PLS1565 was used. Transformants were selected using G418 and confirmed by sequencing. The resulting plasmid (PLS2802) with the 3 genes cloned is depicted in FIG. 3.

Example 2 Transformation of PLS2802 into Yeast and Fermentation

PLS2802 was used to transform S. cerevisiae (THERMOSACC®; LYCC6825; Lallemand). Transformants were selected on YPD agar plates with 200 ug/ml G418 at 30° C. for 48 hrs. Single colonies were used to inoculate 250 μl of defined media (60 g/L glucose; 3 g/L potassium phosphate, 5 g/L ammonium sulphate, 0.5 g/L magnesium sulphate, 100 mM MES pH 5.5) in Axygen 96-well plates. The cultures were grown at 30° C. for 72 hrs with 85% relative humidity. Then, 40 μl of the culture was used to inoculate 360 μl of 3:1 mixture of defined media (60 g/L glucose; 3 g/L potassium phosphate, 5 g/L ammonium sulphate, 0.5 g/L magnesium sulphate, 100 mM MES pH 5.5) to plant (wheat straw) biomass hydrolysates containing xylose in 96-well Costar deep well plates for propagating the cells. The cultures were grown at 30° C. for 40 hrs with 250 rpm shaking. At 40 hrs, the cells were spun down at 22° C. for 10 mins and evaluated for xylose fermentation activity.

For fermentation, cells were re-suspended in 400 ul of plant (wheat straw) biomass hydrolysates containing xylose and the plates were sealed with mats. Plates were incubated at 30° C., with 100 rpm shaking. At different timepoints, cells were harvested and the residual sugars and ethanol in the supernatant were measured by a standard HPLC based method known in the art (See e.g., DuPont et al., Carb. Polym., 68:1-16 [2007], which is incorporated herein by reference). In some experiments, the residual xylose in the supernatant was measured using a spectrophotometric assay (e.g., Megazyme xylose assay; Cat no. K-XYLOSE) performed according to the manufacture's protocol. Strains transformed with the empty vector were used as a control. Strains transformed with the xylose pathway consumed significantly higher amounts of xylose at each of the timepoints tested compared to the vector control.

Example 3 Effect of Codon Optimization on XR, XD Activity

To evaluate the effect of codon optimization, alternate sequences of XR and XD genes were tested. Each of the genes were synthesized with either their native Pichia stiptis sequence (XR.1, XD.1) or with codon optimization towards highly expressed yeast sequences (XR.3; XD.3) (Brat et al Appl. Environ. Microbiol., 75:2304-2311 [2009]). To evaluate the effect of the optimization, XR and XD genes were cloned into vector PLS1448 with different combinations (XR.1-XD.1-XK.2; XR.2-XD.1-XK.2; XR.3-XD.1-XK.2; XR.1-XD.2-XK.2; XR.1-XD.3-XK.2). The variants were used to transform S. cerevisiae (THERMOSACC®; LYCC6469; Lallemand). Transformants were selected on YPD agar plates containing 200 ug/ml G418 at 30° C. for 48 hrs. Single colonies were used to inoculate 250 μl of YPD in Axygen 96-well plates. The cultures were grown at 30° C. for 72 hrs with 85% relative humidity. Then, 40 μl of the culture was used to inoculate 360 μl of YPD with 20 g/L xylose in 96 well Costar deep well plates for propagating the cells. The cultures were grown at 30° C. for 40 hrs with 250 rpm shaking. At 40 hrs, the cells were spun down at 22° C. for 10 mins and evaluated for xylose fermentation activity.

For fermentation, cells were re-suspended in 400 ul of YPD with 20 g/L xylose and the plates were sealed with mats. Plates were incubated at 30° C. at 100 rpm shaking. At 48 hrs, cells were harvested and the residual xylose in the supernatant was measured by a spectrophotometric assay as described above. The best xylose consumption was seen with XR.1-XD.2-XK.2 (PLS6058) variant (See, Table 3-1).

TABLE 3-1 Xylose Consumption by Yeast Strain Transformed With Xylose Pathway Strain Xylose Consumed (g/L) THERMOSACC ® + PLS1448 2.5 (vector control) THERMOSACC ® + XR.2-XD.2-XK.2 12.5 THERMOSACC ® + XR.1-XD.2-XK.2 15.8 THERMOSACC ® + XR.3-XD.2-XK.2 2.5 THERMOSACC ® + XR.2-XD.1-XK.2 13.8 THERMOSACC ® + XR.2-XD.3-XK.2 13.5

Example 4 Construction of XR and XD Variant Libraries

Pichia stipitis xylose reductase and xylitol dehydrogenase were subjected to directed evolution algorithms to improve xylose utilization activity using methods as known in the art (See e.g., WO 2010/144103). Purified PCR products encoding XR and/or XD variants (i.e., comprising amino acid substitutions) and the vector portion of plasmid PLS2802 were pooled and used to transform S. cerevisiae (THERMOSACC®; LYCC6825; Lallemand) to produce a library via homologous recombination (See e.g., Oldenburg et al., Nucl. Acids Res., 25(2):451-452 [1997], which is incorporated herein by reference). The library was screened for xylose fermentation activity as described in Example 2. The xylose utilization was measured relative to the positive control (XR.1-XD.2-XK.2). Strains that had activity significantly above the positive control were retested in replicate and the variants sequenced to identify mutations. The results are provided in the following Tables.

TABLE 4-1 XR and XD Variants Exhibiting Improved Activity Xylose utilization Activity XR XD compared Active Silent Active Silent to WT Variant Mutations wrt Mutations wrt Mutations wrt Mutations P. stipitis No: XR WT XR WT XD WT wrt XD WT XR/XD 1 (WT & 1.0 Positive Control) 2 R276F t811a/c812g/c816t I208R ≧1.0 3 R276F t811a/c812g/c816t F209S/N211K ≧1.0 4 S271G c816t/a826c/a828t I208R/N211K ≧1.0 5 S271G c816t/a826c/a828t I208R/N211S ≧1.0 6 R276F t811a/c812g/c816t I208R/F209S/N211S ≧1.0 7 S271G c816t/a826c/a828t; I208R ≧1.0 8 K270R t811a/c812g/c816t/ I208R/N211R ≧1.0 a826c/a828t 9 K270R t811a/c812g/c816t/ F209S/N211K ≧1.0 a826c/a828t 10  N272P/R276F t811a/c812g F209S/N211K ≧1.0 11  K270R/N272P/R276F t811a/c812g N211K ≧1.0 12  t811a/c812g/c816t/ I208R ≧1.0 a826c/a828t

TABLE 4-2 XR Variants with Improved Activity Xylose Utilization XR-Active XR-Silent Activity Mutations wrt Mutations Compared to WT Variant No: XR WT wrt XR WT P. stipitis XR/XD 1 (WT and 1.00 Positive Control) 13 R276W ≧1.5 14 A266V ≧1.4 15 T232V ≧1.4 16 I267V ≧1.4 17 V255I ≧1.4 18 P2T/A266C ≧1.4 19 S233V ≧1.4 20 K152E ≧1.3 21 L224S ≧1.3 22 A246L ≧1.3 23 a280c/a282t ≧1.3 24 K132N ≧1.3 25 R276M ≧1.3 26 K132A ≧1.3 27 N302S ≧1.3 28 R206S ≧1.3 29 A246S ≧1.3 30 A49G ≧1.3 31 S261N ≧1.2 32 K36Q ≧1.2 33 T232A ≧1.2 34 K155R ≧1.2 35 c438t ≧1.2 36 L224A ≧1.2 37 S261A ≧1.2 38 K152H ≧1.2 39 D134E ≧1.2 40 c906t ≧1.2 41 I62V ≧1.2 42 V283H ≧1.2 43 L108Y ≧1.2 44 c201t ≧1.2 45 D11K ≧1.2 46 S233C ≧1.2 47 D134V ≧1.2 48 S285E ≧1.2 49 A245S ≧1.2 50 S233I ≧1.2 51 S3R ≧1.2 52 I162L ≧1.2 53 c306t ≧1.2 54 G157R ≧1.2 55 T114S ≧1.2 56 L224V ≧1.2 57 t511c/g513t ≧1.2 58 R33L ≧1.2 59 V24G ≧1.2 60 S233K ≧1.2 61 t358c/a360g ≧1.2 62 t82a/c83g ≧1.2 63 K68G ≧1.2 64 N302D ≧1.2 65 D282G ≧1.1 66 R206V ≧1.1 67 a426t ≧1.1 68 G249D ≧1.1 69 t511c/g513t ≧1.1 70 V318C ≧1.1 71 L303V ≧1.1 72 P252C ≧1.1 73 E89V ≧1.1 74 a478c/g480t ≧1.1 75 t703c ≧1.1 76 a378t ≧1.1 77 S97T ≧1.1 78 K155Y c855t ≧1.1 79 N231L ≧1.1 80 N225K ≧1.1 81 N225S ≧1.1 82 S3H ≧1.1 83 A56E ≧1.1 84 c201t ≧1.1 85 K152A ≧1.1 86 P168S ≧1.1 87 D134H ≧1.1 88 N225D ≧1.1 89 Q226D ≧1.1 90 S233G ≧1.1 91 D282C ≧1.1 92 E89N ≧1.1 93 t670c/g672t ≧1.1 94 K281L ≧1.1 95 N302G ≧1.1 96 A297H ≧1.1 97 Q226E ≧1.1 98 S233F ≧1.1 99 K123C ≧1.1 100 S261T ≧1.1 101 K152Q ≧1.1 102 R33V ≧1.1 103 A14V ≧1.1 104 S97R ≧1.1 105 a585g ≧1.1 106 K155D ≧1.1 107 Q226S ≧1.1 108 Q219T ≧1.1 109 I143L ≧1.1 110 D102T; ≧1.1 111 N231H; ≧1.1 112 T232S ≧1.0 113 N225E ≧1.0 114 S184A ≧1.0 115 S3W ≧1.0 116 D282R ≧1.0 117 Q219L ≧1.0 118 T232C ≧1.0 119 R228T ≧1.0 120 K281V ≧1.0 121 S285T ≧1.0 122 t688c ≧1.0 123 N231S ≧1.0 124 c849g ≧1.0 125 K155I ≧1.0 126 N231G ≧1.0 127 K242L ≧1.0 128 t354g ≧1.0 129 A297S ≧1.0 130 K68M ≧1.0 131 c408t ≧1.0 132 t766c ≧1.0 133 T240V ≧1.0 134 E46K/ ≧1.0 D47N 135 I301Y ≧1.0 136 I301C ≧1.0 137 Q226V ≧1.0 138 L303I ≧1.0 139 E279Q ≧1.0 140 P275A ≧1.0 141 D23E/K68R ≧1.0 142 F236L ≧1.0 143 Q219H ≧1.0 144 N225Y ≧1.0 145 N7L ≧1.0 146 A56Y ≧1.0 147 F17W ≧1.0 148 K155A ≧1.0 149 K116Q ≧1.0 150 S261C ≧1.0

TABLE 4-3 Additional XR Variants with Improved Activity Xylose Utilization Activity Variant XR-Active Mutations wrt XR-Silent Mutations wrt (Compared to No. XR WT XR WT Positive Control) 13 (Positive R276W 1.00 Control) 151 P2T/R206S/R276W ≧1.4 152 P2T/A49G/R276W ≧1.4 153 P2T/A49G/R206S/R276W ≧1.4 154 P2T/R276W ≧1.3 155 A49G/R276W ≧1.3 156 P2T/K132N/K152E/R276W ≧1.3 157 P2T/A49G/R276W a280c ≧1.2 158 G227D/R276W t6g ≧1.2 159 A49G/K132A/R206S/R276W ≧1.2 160 P2T/P168S/R276W a853t/g854c/c954g ≧1.2 161 A49G; K152E; R276W a280c ≧1.2 162 R276W c54t ≧1.2 163 P2T/A49G/R276W/V318C a853t/g854c ≧1.2 164 P2T/A49G/K132N/R276W ≧1.2 165 P2T/K132N/R276W a280c ≧1.2 166 P2T/K132N/R276W ≧1.2 167 P2T/G157R/R276W g324a/c402t/a853t/g854c/c954g ≧1.2 168 R33L/A56E/R276W t7a/c8g/g456a ≧1.1 169 A49G/R276W t753c ≧1.1 170 S3H/R33L/A56E/R276W ≧1.1 171 A49G/R276W a280c ≧1.1 172 R276W t82a/c83g/c201t ≧1.1 173 P2T/R276W c954g ≧1.1 174 S3H/T114S/R276W/N302S t699c/a756c ≧1.1 175 P2T/R276W t552c ≧1.1 176 A49G/K132N/R206S/S233V/R276M/ ≧1.1 N302S 177 A49G/K132A/R276W a280c ≧1.1 178 R276W/V318C t6g/t781a/c782g/a853t/g854c ≧1.1 179 P2T/A246L/R276W t781a/c782g/c954g ≧1.1 180 P2T/A49G/K152E/R276W ≧1.1 181 K155R/R206S/R276W ≧1.1 182 R276W g204a ≧1.1 183 R276W a853t/g854c/c954g ≧1.1 184 R276W c201t/a280c ≧1.1 185 T114S/R276W ≧1.1 186 R33L/R276W t7a/c8g ≧1.1 187 R276W c954g ≧1.1 188 P2T/R276W/V318C ≧1.1 189 A49G/K152E/R276W ≧1.1 190 D11K/V255I/R276M g231a/c801t ≧1.1 191 K132N/R276W a280c ≧1.1 192 K52R/R276W ≧1.0 193 P2T/A49G/S233V/A246L/R276W/ ≧1.0 N302S 194 A49G/K132A/K152E/R276W a280c ≧1.0 195 S3H/R276W t99g/c168a ≧1.0 196 P2T/V24G/A49G/R276W c954g ≧1.0 197 R276W c201t ≧1.0 198 R276W/V318C t6g ≧1.0 199 R33L/A56E/R276W t7a/c8g ≧1.0 200 K132N/R276W g465a/t618c/t907c ≧1.0 201 K132N/K155R/R276W/V283H t907c ≧1.0 202 K36Q/R276W ≧1.0 203 R276W a478c ≧1.0 204 R276W c855t ≧1.0 205 R276W t82a/c83g ≧1.0 206 P2T/A49G/S261N/R276W a853t/g854c/c954g ≧1.0 207 P2T/K132A/R276W a280c ≧1.0 208 K132N/R276W ≧1.0 209 R276W t7a/c8g ≧1.0 210 P168S/R276W ≧1.0 211 A56E/T114S/R276W t7a/c8g ≧1.0 212 P2T/A49G/K68G/S261N/R276W/ a853t/g854c ≧1.0 V318C 213 R276W c906t ≧1.0 214 A49G/K132N/K152E/R276W/ a280c ≧1.0 N302S 215 R276W g108a/t907c ≧1.0 216 K132N/R276W t289a/c290g/t291c ≧1.0 217 D11K/R276W g108a/g465a ≧1.0 218 R276W t82a/c83g/c201t/a280c/t358c/ ≧1.0 a378t/t511c 219 P252C/R276W ≧1.0 220 R276W c21t ≧1.0 221 P2T/A49G/R276W/N302S ≧1.0 222 P2T/V24G/R276W/V318C ≧1.0 223 D11K/K155Y/R206S/R276W g108a/t289a/c290g/t291c ≧1.0 224 S3H/R33L/T114S/R276W c168a ≧1.0

TABLE 4-4 Additional XR Variants with Improved Activity Xylose Utilization Activity (Compared Variant XR-Active Mutations wrt XR-Silent Mutations wrt to Positive No: XR WT XR WT Control) 272 P2T/A49G/ t99a/g156a/t747c/t751a/c752g, 1.00 (Positive K132N/S233K/I267V/R276W t753c Control) 377 P2T/E46C/A49G/F107Y/K132N/ t99a/g156a/t747c/t751a/c752g/t753c ≧1.2 S233K/I267V/R276W 378 P2T/C19L/E46C/A49G/F107Y/ t99a/g156a/t747c/t751a/c752g/t753c ≧1.1 K132N/S233K/I267V/R276W 379 P2T/A49G/K132N/S233K/I267V/ t99a/g156a/t552g/t747c/t751a/c752g/ ≧1.1 R276W t753c/a756t/c816t 380 P2T/A49G/K132N/S233K/I267V/ t99a/g108a/g156a/t322c/t747c/t751a/ ≧1.0 R276W c752g/t753c 381 P2T/A49G/K132N/S233K/I267V/ t99a/g156a/t552g/t747c/t751a/c752g/ ≧1.0 R276W t753c 382 P2T/E46C/A49G/K132N/V164I/ t99a/g156a/t747c/t751a/c752g/t753c ≧1.0 S233K/I267V/R276W 383 P2T/C19L/E46C/A49G/K132N/ t99a/g156a/t747c/t751a/c752g/t753c ≧1.0 S233K/ I267V/R276W 384 P2T/A49G/K132N/S233K/R276W t99a/g108a/g156a/t747c/t751a/c752g/ ≧1.0 t753c/c801t 385 P2T/A49G/K132N/P168S/S233K/ t99a/g156a/t322c/t747c/t751a/c752g/ ≧1.0 I267V/R276W t753c 386 P2T/A49G/K132N/P168S/S233K/ t99a/g108a/g156a/t322c/g513a/t747c/ ≧1.0 I267V/R276W t751a/c752g/t753c/c816t

In some additional experiments, the Pichia stipitis xylitol dehydrogenase (XD.2) was subjected to directed evolution to improve xylose utilization activity using PCR amplification known in the art (See e.g., WO 2010/144103). Purified PCR products were pooled and transformed into S. cerevisiae (THERMOSACC®; LYCC6825; Lallemand) to produce the library via homologous recombination (See e.g., Oldenburg et al., Nucl. Acids Res., 25(2):451-452 [1997], which is incorporated herein by reference). The library was screened for xylose fermentation activity as described in Example 2. Variants that exhibited activity significantly above the positive control were retested and sequenced. Variants that exhibited activity improved above background are provided in Table 4-5.

TABLE 4-5 XD Variants with Improved Activity XD-Silent Xylose Utilization XD-Active Mutations Activity Mutations wrt XD (Compared to Variant No: wrt XD WT WT Positive Control) 13 (Positive 1.00 Control) 225 E235K ≧1.2 226 V206A ≧1.2 227 A49Q ≧1.2 228 K352E ≧1.2 229 M215C ≧1.1 230 P256A ≧1.1 231 t24g ≧1.1 232 S81G ≧1.1 233 a768t ≧1.1 234 T19L ≧1.1 235 G202D ≧1.1 236 L189C ≧1.1 237 P5R ≧1.1 238 G231H ≧1.1 239 D210W ≧1.1 240 T230V ≧1.1 241 a780g ≧1.1 242 a732g ≧1.1 243 T286V ≧1.1 244 c630t ≧1.1 245 G231A ≧1.1 246 P5E ≧1.1 247 D218R ≧1.1 248 S228P ≧1.0 249 T19Q ≧1.0 250 V287L ≧1.0 251 V205C ≧1.0 252 V205H ≧1.0 253 V187M ≧1.0 254 H149R ≧1.0 255 F296R ≧1.0 256 A239C ≧1.0 257 L260Q ≧1.0 258 G241W/K307R ≧1.0 259 E235Q ≧1.0 260 D13G ≧1.0 261 A350D ≧1.0 262 T252S ≧1.0 263 C251T ≧1.0 264 N227T ≧1.0 265 M215A ≧1.0 266 C251G ≧1.0 267 K229R ≧1.0 268 F226V ≧1.0 269 P256Q ≧1.0 270 D327A ≧1.0

The following Table (Table 4-6) provides results for XR and XD combinatorial variants with improved activity compared to a positive control having the active mutations indicated in the Table.

TABLE 4-6 XR & XD Combinatorial Variants with Improved Activity Xylose Utilization XR XD Activity Silent Silent Improvement Mutations Active Mutations (Relative to Variant Active Mutations wrt XR Mutations wrt XD Positive No. wrt XR WT WT wrt XD WT WT Control) 152 P2T/A49G/R276W 1.00 (Positive Control) 271 P2T/K132N/ F209S/ ≧1.2 K152E/R276W N211K 272 P2T/A49G/ t99a/g156a/ ≧1.2 K132N/S233K/ t747c/t751a/ I267V/R276W c752g/t753c 273 P2T/A49G/K132N/I267V/ t99a/g204a/ ≧1.2 R276W t618c/t697a/ c698g/t699c/ t747c/t751a/ c752g/t753c 274 P2T/A49G/P168S/R206S/ g456a/t895c ≧1.2 I267V/ R276W 275 P2T/A49G/K52R/K152E/ g231a/t322c ≧1.2 S233K/ I267V/R276W 276 P2T/A49G/ ≧1.2 K132N/I267V/ R276W 277 P2T/A49G/ G232L ≧1.2 R276W 278 P2T/A49G/ t99a/g204a ≧1.2 K132N/R276W 279 P2T/A49G/ t99a/t747c/ ≧1.2 K132N/I267V/ t751a/c752g/ R276W t753c 280 P2T/A49G/ t99g/g108a/ ≧1.2 K132N/R276W a853t/g854c/ c855g 281 P2T/A49G/S233K/I267V/ t99a/g513a/ ≧1.2 R276W t747c/t751a/ c752g/t753c 282 P2T/A49G/R206S/S233K/ t895c ≧1.2 I267V/ R276W 283 P2T/A49G/ g156a/g204a/ ≧1.2 K132N/R276W c801t 284 P2T/A49G/ t753g/a756t/ ≧1.2 K132N/R276W a853t/g854c/ c855g 285 P2T/A49G/S233K/I267V/ t895c ≧1.2 R276W 286 P2T/A49G/K152E/P168S/ t895c ≧1.1 S233V/I267V/R276W 287 P2T/A49G/S233K/I267V/ t618c/t747c/ ≧1.1 R276W t751a/c752g/ t753c 288 P2T/A49G/R276W T225X a693w ≧1.1 289 P2T/A49G/R276W c336t ≧1.1 290 P2T/A49G/S233K/I267V/ t322c/t895c ≧1.1 R276W 291 P2T/A49G/R276W E235K ≧1.1 292 P2T/A49G/S233K/I267V/ t618c/t895c ≧1.1 R276W 293 P2T/A49G/R276W K214A ≧1.1 294 P2T/A49G/K132N/I267V/ t99a/g156a ≧1.1 R276W 295 P2T/A49G/R276W K229V ≧1.1 296 P2T/A49G/R276W ≧1.1 297 P2T/A49G/R206S/ t9c/t895c ≧1.1 S233K/I267V/R276W 298 P2T/A49G/I267V/ t99a/t697a/ ≧1.1 R276W c698g/t699c/ t747c/t751a/ c752g/t753c 299 P2T/A49G/P168S/ t9c/t618c ≧1.1 S233K/R276W 300 P2T/A49G/S233K/ g834a/t895c ≧1.1 I267V/R276W 301 P2T/A49G/G227D/ t753g ≧1.1 P252C/G264D/R276W 302 P2T/A49G/K132N/ a853t/g854c/ ≧1.1 R276W c855g 303 P2T/S3H/A49G/P168S/ g456a/c801t/ ≧1.1 S233K/R276W t895c 304 P2T/A49G/S233K/ t99a/t618c/ ≧1.1 I267V/R276W t747c/t751a/ c752g/t753c 305 P2T/R206S/R276W A49Q ≧1.1 306 P2T/A49G/I267V/ g156a ≧1.1 R276W 307 P2T/A49G/I267V/ t895c/ ≧1.1 R276W 308 P2T/A49G/R206S/ a504g ≧1.1 S233V/R276W 309 P2T/A49G/I267V/ t99a ≧1.1 R276W 310 P2T/A49G/S233K/ c801t/t895c ≧1.1 R276W 311 P2T/A49G/R206S/ t697a/c698g/ ≧1.1 R276W c801t/t895c 312 P2T/A49G/P252C/ t753g/a853t/ ≧1.1 R276W g854c/c855g 313 P2T/K132N/K152E/ K352E ≧1.1 R276W 314 P2T/A49G/I267V/ t747c/t751a/ ≧1.1 R276W c752g/t753c 315 P2T/A49G/I267V/ ≧1.1 R276W 316 P2T/A49G/R276W C251A ≧1.1 317 P2T/A49G/R276W G232R ≧1.1 318 P2T/A49G/R206S/ c801t/t895c ≧1.1 S233V/R276W 319 P2T/A49G/K132N/ g204a/t753g/ ≧1.1 R276W a756t/a853t/ g854c/c855g 320 P2T/A49G/R206S/ t697a/c698g/ ≧1.1 R276W t699c 321 P2T/A49G/R276W K229M ≧1.1 322 P2T/A49G/S233K/ t322c/t618c/ ≧1.1 I267V/R276W t895c 323 P2T/K132N/K152E/ A49Q/E235K ≧1.1 R276W 324 P2T/A49G/R276W a780t ≧1.1 325 P2T/A49G/I267V/ t99a/g156a/ ≧1.1 R276W g204a 326 P2T/A49G/R276W D327X ≧1.1 327 P2T/K132N/K152E/ A49Q ≧1.1 R276W 328 P2T/A49G/P168S/ ≧1.1 R276W 329 P2T/A49G/R276W V206A ≧1.0 330 P2T/A49G/R276W V206A ≧1.0 331 P2T/A49G/R276W K337R ≧1.0 332 P2T/A49G/K152E/ a504g ≧1.0 I267V/R276W 333 P2T/A49G/K132N/ a478c/g480t ≧1.0 R276W 334 P2T/K36Q/A49G/ t99g/t753g/ ≧1.0 G227D/R276W a756t/a853t/ g854c/c855g 335 P2T/A49G/R276W V287L ≧1.0 336 P2T/A49G/D86N/ g204a/a478c/ ≧1.0 K132N/R276W g480t/t753g/ a756t/a853t/ g854c/c855g 337 P2T/A49G/R206S/ A49Q ≧1.0 R276W 338 P2T/A49G/R276W G241E ≧1.0 339 P2T/A49G/G227D/ t753g/a756t/ ≧1.0 R276W a853t/g854c/ c855g 340 P2T/S3X/A49G/S233X/ t84w ≧1.0 I267X/R276W/L299X 341 P2T/A49G/I267V/ a504g/c816t/ ≧1.0 R276W t895c 342 P2T/A49G/R276W H112Q ≧1.0 343 P2T/A49G/R276W K238G ≧1.0 344 P2T/A49G/P168S/ t895c ≧1.0 R276W 345 P2T/A49G/G227D/ ≧1.0 I267V/R276W 346 P2T/A49G/K132N/ t753g/a853t/ ≧1.0 G227D/P252C/R276W g854c/c855g 347 P2T/A49G/R276W K352Q ≧1.0 348 P2T/A49G/I267V/ g204a/t697a/ ≧1.0 R276W c698g/t699c/ t747c/t751a/ c752g/t753c 349 R276F t811a/c812g/ I208R/N211K ≧1.0 c816t 350 P2T/A49G/I267V/ g231a/t322c/ ≧1.0 R276W t895c 351 P2T/A49G/R276W K259S ≧1.0 352 P2T/A49G/K132N/ a478c/g480t/ ≧1.0 G227D/R276W t753g/a756t/ a853t/g854c/ c855g 353 P2T/A49G/I267V/ g156a/t747c/ ≧1.0 R276W t751a/c752g/ t753c 354 P2T/A49G/S233V/ g156a/g231a/ ≧1.0 I267V/R276W t322c/ g456a/t895c 355 P2T/A49G/G227D/ t753g/a853t/ ≧1.0 P252C/R276W g854c/c855g 356 P2T/A49G/R276W K352G ≧1.0 357 P2T/A49G/R276W a853t/g854c/ ≧1.0 c855g 358 P2T/K132N/K152E/ E235K ≧1.0 R276W 359 P2T/A49G/K52R/ t9c/t84a/ ≧1.0 R206S/I267V/R276W g231a/t697a/ c698g/t895c 360 P2T/A49G/K132N/ ≧1.0 R276W 361 P2T/A49G/R276W K352E ≧1.0 362 P2T/A49G/K52R/ t99a ≧1.0 R276W 363 P2T/A49G/R276W H112A ≧1.0 364 P2T/A49G/S233V/ t895c ≧1.0 R276W 365 P2T/R206S/R276W E235K ≧1.0 366 P2T/A49G/R276W t99a/g156a/ ≧1.0 g204a 367 P2T/A49G/R276W S26A ≧1.0 368 P2T/A49G/R276W D210A ≧1.0 369 P2T/A49G/R206S/ K352E ≧1.0 R276W 370 P2T/A49G/R276W t618c/t697a/ ≧1.0 c698g/c801t/ t895c 371 P2T/A49G/S233V/ a504g/t895c ≧1.0 I267V/R276W 372 P2T/W20X/A49G/ c111n/c822t/ ≧1.0 D63X/R276W a825c/a853t/ g854c/c855g 373 P2T/A49G/R276W t84a ≧1.0 374 P2T/A49G/R276W g204a/c801t ≧1.0 375 P2T/A49G/R276W t552g/c801t/ ≧1.0 t895c 376 P2T/A49G/R276W c801t ≧1.0

The sequences of some variants included in the above table(s) are provided below. Variant 3 has active substitutions in both the XR and XD (R276F and F209S/N211K, respectively), as well as XR silent mutations (t811a/c812g/c816t), as shown in SEQ ID NOS:50 and 51 (XR DNA and amino acid sequences, respectively), and SEQ ID NOS:52 and 53 (XD DNA and amino acid sequences, respectively).

(SEQ ID NO: 50) ATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGT TTCGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTAC CGTGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTAC GCCAACGAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAA GGTATCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAAC AACTACCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTT TCTGACTTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCA GTCACCTTCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTC TACTGTGGTAAGGGTGACAACTTCGACTACGAAGATGTTCCAATTTTA GAGACCTGGAAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGG TCTATCGGTGTTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTG AGAGGTGCTACCATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCA TACTTGCAACAACCAAGATTGATCGAGTTCGCTCAATCCCGTGGTATT GCTGTCACCGCTTACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTG AACCAAGGTAGAGCTTTGAACACTTCTCCATTGTTCGAGAACGAAACT ATCAAGGCTATCGCTGCTAAGCACGGTAAGTCTCCAGCTCAAGTCTTG TTGAGATGGTCTTCCCAAAGAGGCATTGCCATCATTCCAAAGAGCAAT ACTGTCCCATTCTTGTTGGAAAACAAGGATGTCAACAGCTTCGACTTG GACGAACAAGATTTCGCTGACATTGCCAAGTTGGACATCAACTTGAGA TTCAACGACCCATGGGACTGGGACAAGATTCCTATCTTCGTCTAA (SEQ ID NO: 51) MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDY ANEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTL SDLQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPIL ETWKALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHP YLQQPRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENET IKAIAAKHGKSPAQVLLRWSSQRGIAIIPKSNTVPFLLENKDVNSFDL DEQDFADIAKLDINLRFNDPWDWDKIPIFV (SEQ ID NO: 52) ATGACCGCTAATCCCTCTCTTGTTTTGAATAAGATTGACGACATTTCT TTTGAAACTTACGATGCTCCCGAAATTAGCGAACCCACAGACGTTTTA GTTCAAGTTAAAAAAACTGGTATCTGCGGTTCTGACATCCACTTCTAC GCTCATGGAAGGATCGGCAACTTCGTCTTAACAAAGCCAATGGTTCTG GGTCATGAAAGCGCGGGTACTGTTGTTCAAGTCGGTAAAGGTGTTACT TCACTGAAGGTTGGTGATAACGTCGCAATCGAGCCCGGTATTCCATCT AGGTTCAGTGATGAGTACAAATCTGGTCACTACAACCTGTGTCCACAC ATGGCATTTGCTGCTACTCCCAATTCTAAAGAGGGTGAACCAAACCCA CCAGGAACTCTATGTAAGTACTTCAAATCTCCAGAAGACTTCCTGGTT AAGTTACCCGATCATGTTTCTTTGGAGTTGGGTGCTTTGGTCGAGCCA CTATCTGTTGGGGTCCATGCTAGTAAATTAGGCTCCGTTGCATTTGGC GATTACGTTGCTGTTTTTGGTGCTGGTCCAGTAGGATTACTGGCTGCC GCTGTCGCTAAGACATTTGGTGCCAAGGGTGTGATTGTCGTTGATATA TCTGACAAGAAGCTGAAGATGGCCAAAGACATAGGTGCCGCTACACAT ACCTTCAACTCCAAGACGGGAGGTAGTGAAGAATTGATCAAAGCCTTC GGTGGTAATGTACCAAATGTTGTCTTGGAATGTACTGGGGCTGAACCA TGTATTAAGCTAGGTGTTGATGCCATCGCACCAGGTGGTAGATTCGTG CAAGTTGGTAATGCTGCTGGTCCCGTGTCCTTTCCCATAACAGTGTTC GCTATGAAAGAACTTACTTTGTTTGGTTCATTTCGTTATGGTTTCAAC GACTATAAGACAGCCGTGGGTATCTTTGATACTAACTACCAGAACGGT AGAGAGAATGCTCCCATTGACTTTGAACAGCTTATCACGCACAGATAC AAATTCAAAGACGCCATTGAAGCCTACGACCTAGTAAGAGCAGGTAAA GGGGCTGTCAAGTGTTTGATTGATGGTCCAGAATAA (SEQ ID NO: 53) MTANPSLVLNKIDDISFETYDAPEISEPTDVLVQVKKTGICGSDIHFY AHGRIGNFVLTKPMVLGHESAGTVVQVGKGVTSLKVGDNVAIEPGIPS RFSDEYKSGHYNLCPHMAFAATPNSKEGEPNPPGTLCKYFKSPEDFLV KLPDHVSLELGALVEPLSVGVHASKLGSVAFGDYVAVFGAGPVGLLAA AVAKTFGAKGVIVVDISDKKLKMAKDIGAATHTFNSKTGGSEELIKAF GGNVPNVVLECTGAEPCIKLGVDAIAPGGRFVQVGNAAGPVSFPITVF AMKELTLFGSFRYGFNDYKTAVGIFDTNYQNGRENAPIDFEQLITHRY KFKDAIEAYDLVRAGKGAVKCLIDGPE

The DNA and amino acid sequences of variant 152 (P2T/A49G/R276W in XR) are provided below (SEQ ID NOS:54 and 55).

(SEQ ID NO: 54) ATGACCTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTT TCGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTACCG TGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGGC AACGAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTA TCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTA CCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGAC TTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCT TCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGG TAAGGGTGACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGG AAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGGTCTATCGGTG TTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTAC CATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAA CCAAGATTGATCGAGTTCGCTCAATCCCGTGGTATTGCTGTCACCGCTT ACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGC TTTGAACACTTCTCCATTGTTCGAGAACGAAACTATCAAGGCTATCGCT GCTAAGCACGGTAAGTCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCC AAAGAGGCATTGCCATCATTCCAAAGTCCAACACTGTCCCATGGTTGTT GGAAAACAAGGATGTCAACAGCTTCGACTTGGACGAACAAGATTTCGCT GACATTGCCAAGTTGGACATCAACTTGAGATTCAACGACCCATGGGACT GGGACAAGATTCCTATCTTCGTCTAA (SEQ ID NO: 55) MTSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDYG NEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSD LQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETW KALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQ PRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIA AKHGKSPAQVLLRWSSQRGIAIIPKSNTVPWLLENKDVNSFDLDEQDFA DIAKLDINLRFNDPWDWDKIPIFV

The DNA and amino acid sequences of variant 272 (P2T/A49G/K132N/S233K/I267V/R276W in XR) are provided below (SEQ ID NOS:56 and 57).

(SEQ ID NO: 56) ATGACCTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGT TTCGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTAC CGAGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTAC GGCAACGAAAAATTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAA GGTATCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAAC AACTACCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTT TCTGACTTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCA GTCACCTTCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTC TACTGTGGTAACGGTGACAACTTCGACTACGAAGATGTTCCAATTTTA GAGACCTGGAAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGG TCTATCGGTGTTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTG AGAGGTGCTACCATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCA TACTTGCAACAACCAAGATTGATCGAGTTCGCTCAATCCCGTGGTATT GCTGTCACCGCTTACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTG AACCAAGGTAGAGCTTTGAACACTAAGCCATTGTTCGAGAACGAAACT ATCAAGGCTATCGCTGCTAAGCACGGCAAGAGCCCAGCTCAAGTCTTG TTGAGATGGTCTTCCCAAAGAGGCATTGCCGTTATTCCAAAGTCCAAC ACTGTCCCATGGTTGTTGGAAAACAAGGATGTCAACAGCTTCGACTTG GACGAACAAGATTTCGCTGACATTGCCAAGTTGGACATCAACTTGAGA TTCAACGACCCATGGGACTGGGACAAGATTCCTATCTTCGTCTAA (SEQ ID NO: 57) MTSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDY GNEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTL SDLQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGNGDNFDYEDVPIL ETWKALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHP YLQQPRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTKPLFENET IKAIAAKHGKSPAQVLLRWSSQRGIAVIPKSNTVPWLLENKDVNSFDL DEQDFADIAKLDINLRFNDPWDWDKIPIFV

The DNA and amino acid sequences of variant 377 (P2T/E46C/A49G/F107Y/K132N/S233K/I267V/R276W in XR) are provided below (SEQ ID NOS:58 and 59).

(SEQ ID NO: 58) ATGACCTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGT TTCGGCTGTTGGAAAGTTGACGTTGACACCTGTTCTGAACAGGTCTAC CGAGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTAC GGCAACGAAAAATTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAA GGTATCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAAC AACTACCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTT TCTGACTTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCA GTCACCTTCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTC TACTGTGGTAACGGTGACAACTTCGACTACGAAGATGTTCCAATTTTA GAGACCTGGAAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGG TCTATCGGTGTTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTG AGAGGTGCTACCATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCA TACTTGCAACAACCAAGATTGATCGAGTTCGCTCAATCCCGTGGTATT GCTGTCACCGCTTACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTG AACCAAGGTAGAGCTTTGAACACTAAGCCATTGTTCGAGAACGAAACT ATCAAGGCTATCGCTGCTAAGCACGGCAAGAGCCCAGCTCAAGTCTTG TTGAGATGGTCTTCCCAAAGAGGCATTGCCGTTATTCCAAAGTCCAAC ACTGTCCCATGGTTGTTGGAAAACAAGGATGTCAACAGCTTCGACTTG GACGAACAAGATTTCGCTGACATTGCCAAGTTGGACATCAACTTGAGA TTCAACGACCCATGGGACTGGGACAAGATTCCTATCTTCGTCTAA (SEQ ID NO: 59) MTSIKLNSGYDMPAVGFGCWKVDVDTCSEQVYRAIKTGYRLFDGAEDY GNEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTL SDLQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGNGDNFDYEDVPIL ETWKALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHP YLQQPRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTKPLFENET IKAIAAKHGKSPAQVLLRWSSQRGIAVIPKSNTVPWLLENKDVNSFDL DEQDFADIAKLDINLRFNDPWDWDKIPIFV

While particular embodiments of the present invention have been illustrated and described, it will be apparent to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the present invention. Therefore, it is intended that the present invention encompass all such changes and modifications with the scope of the present invention.

The present invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part(s) of the invention. The invention described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is/are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation. There is no intention that in the use of such terms and expressions, of excluding any equivalents of the features described and/or shown or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Thus, it should be understood that although the present invention has been specifically disclosed by some preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be utilized by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present invention. 

We claim:
 1. An isolated xylose reductase variant, wherein said variant has xylose reductase activity and comprises a substitution at at least two positions selected from 46, 47, and 49, in SEQ ID NO:2.
 2. The isolated xylose reductase variant of claim 1, wherein said variant has xylose reductase activity and further comprises a substitution at one or more positions selected from P2T, S3H, S3R, S3W, S3X, N7L, D11K, A14V, F17W, C19L, W20X, D23E, V24G, R33L, R33V, K36Q, E46C, E46K, D47G, D47N, A49G, K52R, A56E, A56Y, I62V, D63X, K68G, K68M, K68R, D86N, E89N, E89V, S97R, S97T, D102T, F107Y, L108Y, T114S, K116Q, K123C, K132A, K132N, D134E, D134H, D134V, I143L, K152A, K152E, K152H, K152Q, K155A, K155D, K155I, K155R, K155Y, G157R, I162L, P168S, S184A, R206S, R206V, Q219H, Q219L, Q219T, L224A, L224S, L224V, N225D, N225E, N225K, N225S, N225Y, Q226D, Q226E, Q226S, Q226V, G227D, R228T, N231G, N231H, N231L, N231S, T232A, T232C, T232S, T232V, S233C, S233F, S233G, S233I, S233K, S233V, S233X, F236L, T240V, K242L, A245S, A246L, A246S, G249D, P252C, V255I, S261A, S261C, S261N, S261T, G264D; A266V, A266C, I267V, I267X, K270R, S271G, N272P, P275A, R276M, R276F, R276W, E279Q, K281L, K281V, D282C, D282G, D282R, V283H, S285E, S285T, A297H, A297S, L299X, I301C, I301Y, N302D, N302G, N302S, L303I, L303V, and V318C, in SEQ ID NO:2.
 3. The isolated xylose reductase variant of claim 1, wherein said variant has xylose reductase activity and further comprises at least one of the following substitutions and/or substitution sets P2T/S3H/A49G/P168S/S233K/R276W; P2T/S3X/A49G/S233X/I267X/R276W/L299X; P2T/C19L/E46C/A49G/F107Y/K132N/S233K/1267V/R276W; P2T/C19L/E46C/A49G/K132N/S233K/I267V/R276W; P2T/W20X/A49G/D63X/R276W; P2T/V24G/A49G; P2T/V24G/A49G/R276W; P2T/K36Q/A49G/G227D/R276W; P2T/E46C/A49G/F107Y/K132N/S233K/I267V/R276W; P2T/E46C/A49G/K132N/V164I/S233K/I267V/R276W; P2T/A49G; P2T/A49G/K52R/K152E/S233K/I267V/R276W; P2T/A49G/K52R/R206S/I267V/R276W; P2T/A49G/K52R/R276W; P2T/A49G/K68G/S261N/R276W/V318C; P2T/A49G/K68G/S261N/V318C; P2T/A49G/D86N/K132N/R276W; P2T/A49G/K132N; P2T/A49G/K132N/P168S/S233K/I267V/R276W; P2T/A49G/K132N/G227D/P252C/R276W; P2T/A49G/K132N/G227D/R276W; P2T/A49G/K132N/S233K/I267V/R276W; P2T/A49G/K132N/S233K/R276W; P2T/A49G/K132N/I267V/R276W; P2T/A49G/K132N/R276W; PT2/A49G/K152E; P2T/A49G/K152E/P168S/S233V/I267V/R276W; P2T/A49G/K152E/I267V/R276W; P2T/A49G/K152E/R276W; P2T/A49G/P168S/R206S/I267V/R276W; P2T/A49G/P168S/S233K/R276W; P2T/A49G/P168S/R276W; P2T/A49G/R206S/S233K/I267V/R276W; P2T/A49G/R206S; P2T/A49G/R206S/R276W; P2T/A49G/G227D/P252C/G264D/R276W; P2T/A49G/G227D/P252C/R276W; P2T/A49G/G227D/I267V/R276W; P2T/A49G/G227D/R276W; P2T/A49G/S233K/R276W; P2T/A49G/S233K/I267V/R276W; P2T/A49G/S233V/A246L/R276W/N302S; P2T/A49G/S233V/A246L/N302S; P2T/A49G/S233V/I267V/R276W; P2T/A49G/S233V/R276W; P2T/A49G/P252C/R276W; P2T/A49G/S261N; P2T/A49G/S261N/R276W; P2T/A49G/I267V/R276W; P2T/A49G/R276W; P2T/A46G/R276W/N302S; P2T/A49G/R276W/V318C; P2T/A49G/N302S; P2T/A49G/V319C; E46K/D47N; A49G/K132A; A49G/K132A/K152E; A49G/K132A/K152E/R276W; A49G/K132A/R206S; A49G/K132A/R276W; A49G/K132A/R206S/R276W; A49G/K132N/K152E/R276W; A49G/K132N/K152E/R276W/N302S; A49G/K132N/K152E/N302S; A49G/K132N/R206S/S233V/W276M/N302S; A49G/K132N/R206S/S233V/R276M/N302S; A49G/K152E; A49G/K152E/R276W; A49G/K152E; and A49G/R276W, in SEQ ID NO:2. 